Additive Manufacturing Using Recombinant Collagen-Containing Formulations

Abstract

To provide a process of additive manufacturing of a three-dimensional object that comprises a collagen-based material in at least a portion thereof, a three-dimensional biological object obtained, and a formulation for the additive manufacturing.SOLUTION: A process of additive manufacturing a three-dimensional object comprises dispensing at least one modeling material formulation to sequentially form a plurality of layers in a configured pattern corresponding to a shape of the object, where for at least a portion of the plurality of layers the dispensing is of a modeling material formulation that comprises a plant-derived recombinant human collagen having at least one curable group. The modeling material formulation has a viscosity of 2,000 centipoises or less, or 1,500 centipoises or less, under conditions of zero shear strain rate and 37°C, or under conditions of a shear strain rate of 5 l / sec and room temperature. A concentration of the recombinant human collagen having a curable group is at least 3 mg / mL. Thereby the three-dimensional object is manufactured.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 517,179, filed June 9, 2017, the entire contents of which are incorporated herein by reference.

[0002] The present invention, in some embodiments thereof, relates to additive manufacturing, and more particularly, but not exclusively, to three-dimensional (3D) bioprinting of 3D objects using collagen-based building materials. [Background technology]

[0003] Collagen is the main component of connective tissue and the most abundant protein in mammals, accounting for about 30% of the protein present in the body. Collagen serves as the main component and major structural-mechanical determinant of most tissue extracellular matrices (ECM) [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].

[0004] Due to its unique characteristics and diverse profile in human body functions, collagen is often chosen among various biocompatible materials to repair tissues, support structural integrity, induce cell infiltration, and promote tissue regeneration. Of the five major collagen types, type I collagen is the most abundant form in the human body. Collagen's unique properties make it a preferred choice for regenerative medicine products.

[0005] Additive manufacturing (AM) is generally the process of producing three-dimensional (3D) objects using a computer model of the object. The basic operation of an AM system consists of slicing the 3D computer model into thin cross sections, converting the results into 2D positional data, and feeding the data to a controller that produces the 3D structure layer by layer.

[0006] A variety of AM techniques exist, including stereolithography, digital light processing (DLP), and three-dimensional (3D) printing (such as 3D inkjet printing). Such techniques generally involve the layer-by-layer deposition and curing (e.g., solidification) of one or more build materials, which typically include a photopolymerizable (light-curable) material.

[0007] For example, stereolithography is an additive manufacturing process that uses a liquid ultraviolet (UV) curable build material and a UV laser. In such a process, a laser beam traces the cross section of the part pattern onto the surface of the dispensed liquid build material at each dispensed layer of the build material. Exposure to the UV laser light causes the traced pattern in the build material to harden and bond to the layer below. After building, the formed part is immersed in a chemical bath to remove excess build material and then cured in a UV oven.

[0008] For example, in a three-dimensional printing process, a build material is dispensed from a dispensing head having a set of nozzles to deposit layers onto a support structure, and depending on the build material, the layers can then be cured or solidified using an appropriate device.

[0009] The build materials may include a modeling material formulation and a support material formulation that upon solidification respectively form the object and a temporary support structure that supports the object being built.

[0010] Modeling material formulations are deposited to produce the desired object, and support material formulations, with or without modeling material elements, are also used to provide support structures in specific areas of the object being built to ensure proper vertical alignment of subsequent object layers, for example, when the object includes overhangs or shapes (e.g., curved shapes, negative angles, gaps, etc.).

[0011] Both the modeling material and the support material are preferably liquid at the working temperature at which they are dispensed, and then solidify upon exposure to a hardening or curing condition, typically a curing energy (e.g., UV curing), to form the required layer shape. After printing is complete, the support structures (if present) are removed to reveal the final shape of the fabricated 3D object. Solidifying (curing) the dispensed materials typically involves polymerization (e.g., photopolymerization) and / or crosslinking (e.g., photocrosslinking).

[0012] The first biological application of additive manufacturing was the creation of three-dimensional sacrificial resin molds to fabricate 3D scaffolds from biological materials.

[0013] 3D bioprinting is an additive manufacturing method that uses biological materials, optionally combined with chemicals and / or cells, to print 3D structures layer by layer, with precise positioning and tight control over the placement of functional components.

[0014] Three-dimensional (3D) bioprinting is gaining momentum in many medical applications, particularly in regenerative medicine, to address the need for complex scaffolds, tissues and organs suitable for transplantation.

[0015] An inherent problem with 3D printing in general is that the mechanical properties of the print medium (the dispensed build material) can differ significantly from the cured (solidified) material after printing.

[0016] To allow for precise control of post-printing curing (e.g., polymerization), build materials typically contain polymerizable (e.g., photopolymerizable) moieties or groups that polymerize (e.g., by chain extension and / or crosslinking) upon dispensing, thereby preserving the geometric shape and imparting the desired physical properties to the final product.

[0017] Various techniques have been developed for 3D bioprinting, including 3D inkjet printing, extrusion printing, laser printing and projection stereolithography [see, for example, Murphy SV, Atala A, Nature Biotechnology. 2014 32(8).; Miller JS, Burdick J. ACS Biomater. Sci. Eng. 2016, 2, 1658-1661]. Each technique has different requirements for the dispensed build material (also referred to herein as the print medium), which stem from the specific application mechanism and the curing / gelling process required to maintain the 3D structure of the scaffold after printing.

[0018] For all techniques, the most important parameters that determine the accuracy and efficiency of printing are the static and dynamic properties of the dispensed build material, e.g., viscosity, shear thinning, and thixotropic properties. The static and dynamic properties of the build material are not only important for printing techniques, but also when considering printing with cells, i.e., cells within the build material dispensed during printing. In this case, the shear forces applied to the build material during printing (dispensing) have a significant impact on cell survival. Therefore, it is desirable to have good control over the specific properties of the print medium over a wide range of conditions, i.e., concentration, temperature, ionic strength, and pH.

[0019] Type I collagen is considered an ideal candidate for use as the main component of the build material in 3D bioprinting.

[0020] Collagen methacrylate can be used as a rapidly self-assembling type I collagen to form crosslinked hydrogels for tissue engineering [see, for example, Isaacson et al., Experimental Eye Research 173, 188-193 (2018)]. Collagen methacrylate has been used with mesenchymal stem cells [Kathryn E. Drzewiecki et al., A thermoreversible, photocrosslinkable collagen bio-ink for free-form fabrication of scaffolds for regenerative medicine, TECHNOLOGY (2017)], fibroblasts, adipose-derived stem cells, epithelial cells, and many more cells. Collagen methacrylate is useful for forming scaffolds of various stiffness by varying collagen concentration and curing conditions (e.g., irradiation intensity and duration).

[0021] Tissue-derived collagen methacrylate has been extensively characterized for its utility in 3D bioprinting (extrusion, inkjet and photolithography) [Drzewiecki, KE et al. Langmuir 30, 11204-11211 (2014); Gaudet, ID & Shreiber, DI Biointerphases 7, 25 (2012)].

[0022] Despite the great advantages of this natural polymer, a number of factors prevent 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 spontaneously forms gels above 20 °C under physiological conditions [see, for example, PureCol, Advanced BioMatrix, Inc.]. The typical temperature-dependent gel formation of tissue-extracted collagen severely hinders accurate flow during printing. A possible solution to this phenomenon is to keep the print medium at a low temperature until application, but this represents a significant technical limitation. An alternative solution is the use of gelatin, a denatured form of collagen that does not gel under these conditions. However, gelatin lacks the true interaction with tissues and cells that natural collagen exhibits, thus missing important biological functions.

[0023] The assignee has developed technology that allows for the purification of native human type I collagen (rhcollagen) by introducing five human genes encoding heterotrimeric type I collagen into tobacco plants [see, e.g., Stein H. (2009) Biomacromolecules; 10:2640-5]. The protein is purified to homogeneity by a cost-effective industrial process that exploits the unique properties of collagen. See also WO2006 / 035442, WO2009 / 053985, WO2011 / 064773, WO2013 / 093921, WO2014 / 147622, ​​and patents and patent applications derived therefrom, all of which are incorporated herein by reference in their entirety. Further background art includes U.S. Patent Application Serial No. 15 / 867,783. Summary of the Invention

[0024] According to one aspect of some embodiments of the present invention, there is provided a formulation for use or usable for additive manufacturing of three-dimensional objects comprising at least in part a collagen-based material, the formulation comprising recombinant human collagen having at least one hardenable group.

[0025] According to some embodiments of the present invention, at least one curable group is a photocurable group.

[0026] According to some embodiments of the invention, the recombinant human collagen is recombinant human type I collagen.

[0027] According to some embodiments of the present invention, the recombinant human collagen is a plant-derived recombinant collagen.

[0028] According to some embodiments of the present invention, the formulation further comprises an aqueous carrier.

[0029] According to some embodiments of the present invention, the formulation has a pH in the range of about 6 to about 8.

[0030] According to some embodiments of the present invention, the aqueous carrier comprises a phosphate buffer.

[0031] According to some embodiments of the present invention, the aqueous carrier comprises an acid.

[0032] According to some embodiments of the present invention, the aqueous carrier comprises a culture medium.

[0033] According to some embodiments of the present invention, the concentration of recombinant human collagen in the modeling material formulation ranges from 0.5 mg / mL to 50 mg / mL or from 0.5 mg / mL to 20 mg / mL.

[0034] According to some embodiments of the present invention, the formulation further comprises at least one hardenable material other than recombinant human collagen having hardenable groups.

[0035] According to some embodiments of the present invention, the weight ratio of recombinant human collagen having curable groups to the at least one curable material ranges from 10:1 to 1:2, alternatively from 10:1 to 1:1, or alternatively from 5:1 to 2:1.

[0036] According to some embodiments of the invention, the formulation further comprises an agent that modifies the mechanical and / or rheological and / or physical properties of the formulation and / or parts of an object constructed with said formulation.

[0037] According to some embodiments of the present invention, the formulation further comprises a biological material other than human recombinant collagen.

[0038] According to some embodiments of the present invention, the formulation is a shear thinning formulation.

[0039] According to some embodiments of the present invention, the formulation is a heat thinning formulation.

[0040] According to some embodiments of the invention, the formulation has a viscosity of 2,000 centipoise or less, or 1,500 centipoise or less, at zero shear strain rate and 37° C., and a concentration of recombinant human collagen having hardenable groups of at least 3 mg / mL.

[0041] According to some embodiments of the present invention, the modeling material formulation has a viscosity of 2,000 centipoise or less, or 1,500 centipoise or less, at a shear strain rate of 5 1 / sec and room temperature, and a concentration of recombinant human collagen having hardenable groups of at least 3 mg / mL.

[0042] According to some embodiments of the present invention, the formulation has a storage modulus (G') of at least 1,000 Pa when solidified.

[0043] According to some embodiments of the present invention, the formulation exhibits at least a 10-fold increase in its storage modulus (G') upon solidification.

[0044] According to one aspect of some embodiments of the present invention, there is provided a process or method for additive manufacturing of a three-dimensional object, at least a portion of which is characterized by a collagen-based material, comprising dispensing at least one modeling material formulation to successively form a plurality of layers in a configuration pattern corresponding to a shape of the object, wherein for at least a portion of the plurality of layers, the dispensing is a modeling material formulation comprising recombinant human collagen having at least one hardenable group as described in each embodiment and any combination thereof herein, thereby providing a process for producing a three-dimensional object.

[0045] According to some of the embodiments described herein, the process further comprises exposing a portion of the plurality of layers to curing conditions suitable for solidifying the recombinant human collagen having at least one curable group.

[0046] According to some of the embodiments described herein, at least one curable group is a photocurable group.

[0047] According to some of the embodiments described herein, the process further includes exposing a portion of the plurality of layers to radiation.

[0048] According to some of the embodiments described herein, the recombinant human collagen is recombinant human type I collagen.

[0049] According to some of the embodiments described herein, for at least a portion of the plurality of layers, the dispensing further comprises dispensing a modeling material formulation comprising at least one hardenable material other than recombinant human collagen having hardenable groups.

[0050] According to some of the embodiments described herein, the dispensing is of a modeling material formulation that includes recombinant human collagen having hardenable groups and at least one hardenable material.

[0051] According to some of the embodiments described herein, the weight ratio of the recombinant human collagen having hardenable groups to the at least one hardenable material ranges from 10:1 to 1:2, or from 10:1 to 1:1, or from 5:1 to 2:1.

[0052] According to some of the embodiments described herein, the at least one hardenable material includes hyaluronic acid having at least one hardenable group.

[0053] According to some of the embodiments described herein, the modeling material formulation is a further dispenser of a modeling material formulation that includes reagents that modify the mechanical and / or rheological and / or physical properties of the formulation and / or portions of an object constructed with the formulation.

[0054] According to some of the embodiments described herein, the dispensing is a further dispensing of a modeling material formulation that includes a reagent that modifies the mechanical and / or rheological and / or physical properties of the formulation and / or portions of the object constructed from the formulation.

[0055] According to some of the embodiments described herein, the agent is a thixotropic agent.

[0056] According to some of the embodiments described herein, the reagent is a gel forming agent.

[0057] According to some of the embodiments described herein, the modeling material formulation further comprises a biological material other than human recombinant collagen.

[0058] According to some of the embodiments described herein, in at least a portion of the plurality of layers, the dispensing is a further dispensing of a modeling material formulation that includes a biological material other than human recombinant collagen.

[0059] According to some of the embodiments described herein, the modeling material formulation comprising human recombinant collagen further comprises a biological material.

[0060] According to some embodiments of the invention, the temperature at which dispensing is carried out is at least 10°C, or at least 20°C, or 37°C.

[0061] According to an aspect of some embodiments of the present invention, there is provided a three-dimensional biological object obtained by the process of AM of each of the embodiments described herein and any combination thereof.

[0062] According to some embodiments of the present invention, the object further comprises in or on at least a portion thereof a biological material other than the collagen-based material.

[0063] According to some embodiments of the present invention, the article is used or usable for repairing damaged tissue.

[0064] According to some embodiments of the invention, the object is an artificial tissue or organ, or is for the construction or usable for the construction of an artificial organ or tissue.

[0065] According to one aspect of some embodiments of the present invention, there is provided a composition comprising a recombinant human collagen having at least one hardenable group as described in each embodiment and any combination thereof herein.

[0066] According to some embodiments of the invention, the composition is for, or can be used to prepare, a modeling material formulation that can be used for additive manufacturing of three-dimensional objects that comprise at least in part a collagen-based material.

[0067] According to some embodiments of the present invention, the composition further comprises an aqueous solution of recombinant human collagen having at least one hardenable group.

[0068] According to one aspect of some embodiments of the present invention, there is provided a kit containing a composition described herein packaged in a kit specific for preparing a modeling material formulation that can be used for additive manufacturing of three-dimensional objects comprising at least in part a collagen-based material.

[0069] According to some embodiments of the present invention, the modeling material formulation further comprises an aqueous carrier, and the kit further comprises instructions for preparing the aqueous carrier or a formulation comprising the aqueous carrier.

[0070] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. Additionally, the materials, methods, and examples are merely illustrative and are not necessarily intended to be limiting.

[0071] In implementing the method and / or system of the present invention, the selection tasks may be performed or completed manually, automatically, or a combination thereof. Furthermore, depending on the actual device or apparatus of the method and / or system of the present invention, some selection tasks may be performed by hardware, software or firmware, or a combination thereof using an operating system.

[0072] For example, hardware for performing the selection task according to embodiments of the present invention may be implemented as a chip or circuit. In the case of software, the selection task according to embodiments of the present invention may be implemented as a number of software instructions executed by a computer using any suitable operating system. In an exemplary embodiment of the present invention, one or more tasks according to exemplary embodiments of the methods and / or systems described herein are performed by a data processor, such as a computing platform executing a number of instructions. Optionally, the data processor includes volatile memory for storing instructions and / or data, and / or non-volatile storage, such as a magnetic hard disk and / or removable media, for storing instructions and / or data. Optionally, a network connection is also provided. Optionally, a display and / or a user input device (e.g., a keyboard and / or a mouse) are also provided.

[0073] Certain embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings, in which it is emphasized that the details shown hereinafter, with particular reference to the drawings, are by way of example and for the purpose of providing a detailed description of embodiments of the invention, in which: [Brief description of the drawings]

[0074] [Figure 1] Figure 1 shows the 1H nuclear magnetic resonance (NMR) spectra of rh collagen (top) and rh collagen methacrylate (bottom) recorded with quantitative analysis using a 500 MHz spectrometer. The signals of the methyl functional group of the methacrylate and the acrylic protons introduced into rh collagen methacrylate are shown in (b) and (c), respectively, and the lysine methylene signal of rh collagen is shown in (a). [Diagram 2]FIG. 2 is a comparative plot showing the viscosity as a function of shear rate for a 2.7 mg / mL solution of bovine collagen in DPBS (solid line) and a 2.79 mg / mL solution of rh collagen in DPBS (dashed line) measured at 4° C. (blue) and 37° C. (red). [Diagram 3] Figure 3 is a comparative plot showing the viscosity as a function of shear rate of a FB solution of 3.4 mg / mL rh collagen measured at 4 °C (blue) and 37 °C (red), showing identical shear thinning behavior at both temperatures. [Figure 4] FIG. 4 is a comparative plot showing the viscosity as a function of shear rate of a 10 mg / mL solution of rhcollagen-MA in DPBS measured at 4° C. (blue) and 37° C. (red), demonstrating shear-thinning behavior at both temperatures. [Diagram 5] Figure 5 is a comparative plot showing the viscosity as a function of increasing temperature of a 10 mg / ml solution of rh collagen-MA in 20 mM acetic acid (blue) and an equivalent solution of bovine collagen methacrylate ink (bovine type I collagen methacrylate) (red), measured with a rheometer using the rotational temperature step CR mode at a shear rate of 5 [l / s]. [Figure 6] 6A-B are bar graphs showing the storage and loss moduli and tan phase shift angle (tan delta) of rh collagen-MA formulations at various concentrations before (FIG. 6A) and after (FIG. 6B) photocuring. [Figure 7] FIG. 7 is a bar graph showing the G′ and G″ values ​​of cured bodies prepared from various formulations of fibrillar rhcollagen-MA recorded in a frequency sweep test at 37° C. and plotted at 1 Hz. [Figure 8] FIG. 8 is a plot showing the G′ and complex viscosity values ​​measured over time for a cured body prepared from a 15 mg / ml solution of rh collagen-MA in 10 mM HCl, recorded with a rheometer using a strain of 7% and an angular frequency of oscillation of 5 rad / sec. [Figure 9]FIG. 9 is a comparative plot showing the viscosity as a function of shear rate for solutions of rhcollagen-MA, rhcollagen-MA plus hyaluronic acid (HA), or rhcollagen-MA and hyaluronic acid-MA (HAMA) in DMEM. [Figure 10] Figure 10 is a comparative plot showing the viscosity as a function of shear rate for a 5 mg / ml rhcollagen-MA solution (col, black curve) and a 5 mg / ml solution of rhcollagen-MA and polyvinyl alcohol methacrylate (PVAMA, green curve) with rhcollagen-MA:PVAMA weight ratios of 5:1 (green solid curve), 2:1 (green dashed curve), or 1:2 (green dotted curve). All solutions were prepared in 0.1 M PB + 11.3 mM NaCl, pH 7.4. [Figure 11] 11 is a comparative plot showing the viscosity as a function of shear rate for a 5 mg / ml rhcollagen-MA solution (col, black curve) and a 5 mg / ml solution of rhcollagen-MA and HAMA (blue curve) with rhcollagen-MA:HAMA weight ratios of 5:1 (blue solid curve) or 2:1 (blue dashed curve). All solutions were prepared in 0.1 M PB + 11.3 mM NaCl, pH 7.4. [Figure 12] Figure 12 is a comparative plot showing the viscosity as a function of shear rate for a 5 mg / ml rhcollagen-MA solution (col, black curve) and a 5 mg / ml solution of rhcollagen-MA and TiO2 (red curve) with rhcollagen-MA:TiO2 weight ratios of 5:1 (red solid curve), 2:1 (red dashed curve), or 1:2 (red dotted curve). All solutions were prepared in 0.1 M PB + 11.3 mM NaCl, pH 7.4. [Figure 13] 13 is a comparative plot showing the viscosity as a function of shear rate for a 5 mg / ml rh collagen-MA solution (col, black curve) and a 5 mg / ml solution of rh collagen-MA and oxidized cellulose (orange curve) with rh collagen-MA:oxidized cellulose weight ratios of 5:1 (orange solid curve), 2:1 (orange dashed curve), or 1:2 (orange dotted curve). All solutions were prepared in 0.1 M PB + 11.3 mM NaCl, pH 7.4. [Figure 14]FIG. 14 shows photographs of cured bodies (scaffolds) obtained from formulations containing rhcollagen-MA and various additives at a weight ratio of rhcollagen-MA:additive of 2:1. [Figure 15] FIG. 15 shows a plot of viscosity as a function of shear rate for 10 mg / ml rhcollagen-MA before curing in 0.1 M phosphate buffer / 0.016 M NaCl (blue) and 10 mM HCl (red) recorded with a rheometer using rotational CR mode at room temperature, showing a more pronounced shear thinning behavior for the PBS solution. [Figure 16] FIG. 16 is a bar graph showing G′ values ​​of hardened objects of 10 mg / ml rhcollagen-MA in 0.1 M phosphate buffer / 0.016 M NaCl and 10 mM HCl recorded with a rheometer using oscillatory CR mode at 37° C. [Figure 17] Figure 17 shows a photograph of a scaffold prepared from rhcollagen-MA solution incorporated into a mouse skin patch (ex vivo) and obtained after transdermal white LED torch irradiation. The skin patch was irradiated on the outside and the rhcollagen-MA solution was dispensed into the subcutaneous mold. [Figure 18] FIG. 18 is a bar graph showing the proliferation of normal human dermal fibroblasts seeded onto scaffolds formed with a solution containing rhcollagen-MA and calcium phosphate particles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] The present invention, some embodiments of which relate to additive manufacturing, and more particularly, but not exclusively, to 3-dimensional (3D) bioprinting of 3D objects using collagen-based building materials.

[0076] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components and / or methods illustrated in the following description and / or drawings and / or examples. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0077] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details exemplified in the following description or examples. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0078] The present inventors have surprisingly found that recombinant human collagen (rhcollagen) is far more resistant to conditions of temperature and ionic strength than tissue-extracted collagen. The present inventors have found that aqueous solutions of rhcollagen do not form gels at temperatures above 20° C., even at 37° C., and that their viscosity is hardly affected by various conditions, such as acidic conditions or salt concentrations above 50 mM.

[0079] Figures 2 and 3 show that the viscosity profiles are similar when the shear rate of the rh collagen solutions is increased at different temperatures. Figure 2 further shows that for the aqueous solutions of tissue-extracted collagen, the viscosity profiles observed at different temperatures are different, with the viscosity of the tissue-extracted collagen solution being substantially higher, especially at low shear rates.

[0080] The inventors have found that the above-mentioned properties of rh-collagen can be advantageously utilized in additive manufacturing processes (e.g., 3D bioprinting) to avoid the need for cooling the additive manufacturing system and to allow better control over the flow properties of the modeling material formulation while allowing it to be performed under conditions compatible with biological additives (e.g., physiological temperature, ionic strength, application of low shear forces).

[0081] While reducing the invention to practice, the inventors have succeeded in generating hardenable groups (e.g., (meth)acrylic groups) as substituents on amino acid residues (e.g., lysine residues) of rh collagen, and have shown that over 80% of the lysine residues are modified to have a methacrylamide group in their side chain rather than an amine group, as shown in FIG. 1 and Table 1.

[0082] Rh collagen having one or more (preferably multiple) curable groups such as (meth)acrylic groups as described herein is also referred to interchangeably throughout this specification as "curable rh collagen" or simply "curable collagen."

[0083] Aqueous solutions of the curable rh collagen were found to exhibit similar relatively low viscosities at low shear rates and similar shear thinning behavior, as shown in Figure 4. Additionally, as shown in Figure 5, the curable rh collagen solutions were found to exhibit thermal thinning behavior within the temperature range of working temperatures that can be used in additive manufacturing processes, exhibiting suitable viscosities in this temperature range, in contrast to the thermal thickening behavior and high viscosity exhibited by the curable tissue-extracted collagen solutions.

[0084] As shown in Figures 6A-B, 7 and 8, the curable rh collagen solutions were further found to exhibit good curability and curing stability, resulting in cured materials with desirable mechanical properties.

[0085] As shown in Figures 10-16, the advantageous properties of the curable rh collagen were found to be maintained in the presence of various additives and when used in different solutions (aqueous carriers).

[0086] The curable rh collagen performed well in an in vitro model of transdermal application, as shown in FIG. 17, and was found to be biocompatible, as shown in FIG.

[0087] Accordingly, embodiments of the present invention relate to compositions comprising curable recombinant human collagen, formulations comprising curable recombinant human collagen, and additive manufacturing processes utilizing the formulations to produce three-dimensional (3D) objects featuring collagen-based materials.

[0088] Additive Manufacturing: According to one aspect of some embodiments of the present invention, a process for additive manufacturing (AM) of a three-dimensional object is provided. According to embodiments of this aspect, the method forms an object by sequentially forming a plurality of layers in a configuration pattern corresponding to the shape of the object. According to embodiments of this aspect, each layer is formed by dispensing at least one uncured build material and exposing the dispensed build material to curing conditions to form a solidified (hardened) material.

[0089] Throughout this specification, the phrase "build material" includes the phrase "uncured build material" or "uncured build material formulation," which is a collective term for materials dispensed in successive layers as described herein. This phrase includes the uncured materials that form the final object, i.e., one or more uncured modeling material formulations, and in some cases also includes the uncured materials used to form the support, i.e., uncured support material formulations. Build materials also include non-curing materials that preferably do not undergo (or will not undergo) any change during the process, such as biological materials or components (other than the curable collagen described herein) and / or other reagents or additives described herein.

[0090] The build material that is dispensed to form successive layers as described herein is also referred to interchangeably herein as a "print medium" or "bioprinting medium."

[0091] The uncured build material can include one or more modeling material formulations, and can be dispensed such that different portions of the object are formed upon solidification (e.g., curing) of the different modeling formulations. Thus, different portions of the object are formed with different solidified (e.g., hardened) modeling materials, or different mixtures of solidified (e.g., hardened) modeling materials.

[0092] In the method of the present embodiment, a three-dimensional object is fabricated layer by layer by forming multiple layers in a configuration pattern that corresponds to the shape of the object.

[0093] Each layer is formed by an additive manufacturing device that scans the two-dimensional surface to pattern it. During the scan, for each target location or group of target locations, the device directs the device to multiple target locations in the two-dimensional layer or surface and determines according to a preset algorithm whether that target location or group of target locations is occupied with build material and what type of build material to deliver thereto. This determination is made according to a computer image of the surface.

[0094] When AM is performed by 3D inkjet printing, uncured build material as defined herein is dispensed from a dispensing head with a set of nozzles, depositing the build material in layers onto a support structure. Thus, the AM device dispenses the build material at target locations to be occupied, leaving other target locations free. The device typically has multiple dispensing heads, each configured to dispense a different build material (e.g., different modeling material formulations (each containing a different biological component, or a different curable material, or a different concentration of curable material) and / or a different support material formulation). Thus, different target locations can be occupied with different build materials (e.g., modeling formulations and / or support formulations as defined herein).

[0095] The final three-dimensional object is formed from the solidified modeling material, or a combination of the solidified modeling material, or a combination of the solidified modeling material and a support material, or modifications thereof (e.g., after curing), all of which are well known to those skilled in the art of additive manufacturing (also known as solid freeform molding).

[0096] In some exemplary embodiments of the invention, an object is manufactured by dispensing a build material comprising two or more different modeling material formulations, with each modeling material formulation being dispensed from a different dispensing head of the AM device. The multiple modeling material formulations are deposited in layers, as needed, preferably during the same pass of the dispensing head. The modeling material formulations and / or combinations of formulations within a layer are selected according to the desired properties of the object.

[0097] An exemplary process according to some embodiments of the present invention begins by receiving 3D printing data corresponding to the shape of an object. The data can be received, for example, from a host computer transmitting digital data related to manufacturing instructions based on computer object data, which can be in the form of, for example, Standard Tessellation Language (STL) or Stereo Lithography Contour (SLC) format, Virtual Reality Modeling Language (VRML), Additive Manufacturing File (AMF) format, Drawing Interchange Format (DXF), Polygon File Format (PLY), Digital Imaging and Communications in Medicine (DICOM), or any other format suitable for computer-aided design (CAD).

[0098] The process continues by dispensing build material, as described herein, layer by layer onto a receiving medium using one or more dispensing (eg, print) heads according to print data.

[0099] Depending on the additive manufacturing method used and the configuration selected, dispensing can be done in the form of droplets or a continuous stream.

[0100] The receiving medium can be a tray of a printing system, or a support or medium formed or coated with a biocompatible material (e.g., a support medium or article commonly used in bioprinting), or a pre-deposited layer.

[0101] In some embodiments, the receiving medium includes a sacrificial hydrogel or other biocompatible material as a mold for embedding the object, which is then removed by chemical, mechanical, or physical (e.g., heating or cooling) means. Such sacrificial hydrogels can be formed, for example, of pluronic materials or gelatin.

[0102] Once the uncured build material has been dispensed onto the receiving medium according to the 3D data, the dispensed formulation is then preferably solidified, if necessary. In some embodiments, the next step in the process is to expose the deposited layer to a curing condition. Preferably, the application of the curing condition to each layer occurs after the deposition of this layer and before the deposition of the previous layer.

[0103] As used herein, the term "curing" refers to the process by which a formulation solidifies. The solidification of a formulation is typically accompanied by an increase in the viscosity of the formulation and / or an increase in the storage modulus (G') of the formulation. In some embodiments, a formulation dispensed as a liquid solidifies to a solid or semi-solid (e.g., a gel). A formulation dispensed as a semi-solid (e.g., a soft gel) solidifies to a solid or to a harder or stronger semi-solid (e.g., a strong gel).

[0104] The term "curing" as used herein includes, for example, polymerization of monomeric and / or oligomeric materials and / or crosslinking of polymer chains (crosslinking of polymers present prior to curing or crosslinking of polymeric materials formed upon polymerization of monomers or oligomers). Thus, the product of a curing reaction is typically a polymeric and / or crosslinked material. As used herein, the term also includes partial curing, e.g., at least 20% or at least 30% or at least 40% or at least 50% or at least 60% or at least 70% curing of the formulation, as well as 100% curing of the formulation.

[0105] The phrases "conditions affecting cure" or "conditions for inducing cure", also referred to herein interchangeably as "cure conditions" or "cure-inducing conditions", refer to conditions that, when applied to a formulation containing a curable material, induce cure as defined herein. Such conditions can include, for example, the application of cure energy to the curable material, as described below, and / or contact of the curable material with chemically reactive components (e.g., catalysts, cocatalysts, and activators).

[0106] When the conditions that induce curing include the application of curing energy, the phrase "expose to curing conditions" and grammatical variations thereof refer to exposing the dispensed layer to curing energy, which exposure is typically achieved by applying curing energy to the dispensed layer.

[0107] "Curing energy" typically includes the application of radiation or the application of heat.

[0108] The radiation can be electromagnetic radiation (e.g., ultraviolet or visible light), or electron beam radiation, or ultrasonic radiation, or microwave radiation, depending on the material to be cured. The application (or exposure) of radiation is by a suitable radiation source. For example, as described herein, an ultraviolet source, or a visible light source, or an infrared source, or a xenon light source, or a mercury light source, or a lamp light source, or an LED light source can be used.

[0109] Curable materials or systems that harden upon exposure to radiation are referred to interchangeably herein as "photopolymerizable" or "photoactivatable" or "photohardenable."

[0110] When the curing energy includes heat, curing is also referred to herein and in the art as "thermal curing" and involves the application of thermal energy. As described herein, the application of thermal energy can be accomplished, for example, by heating the receiving medium into which the layer is dispensed or a chamber containing the receiving medium. In some embodiments, heating is accomplished using a resistive heater.

[0111] In some embodiments, the heating is achieved by irradiating the dispensed layer with thermally inducing radiation, which can be achieved, for example, by an IR lamp or a xenon lamp that is operable to emit radiation onto the deposited layer.

[0112] In some embodiments, heating is accomplished by infrared radiation from a ceramic lamp, for example a ceramic lamp that provides infrared radiation at about 3 μm to about 4 μm (eg, about 3.5 μm).

[0113] Curable materials or systems that harden upon exposure to heat are referred to herein as "thermosetting" or "thermoactivatable" or "thermopolymerizable."

[0114] In some of the embodiments described herein, solidifying the dispensed formulation comprises exposing the dispensed formulation to curing conditions, e.g., irradiation (illumination), as described in any of the embodiments herein.

[0115] In some embodiments, exposure to curing conditions occurs for a short period of time, e.g., less than 3 minutes, less than 300 seconds, e.g., from 10 seconds to 240 seconds, or from 10 seconds to 120 seconds, or from 10 seconds to 60 seconds, including any intermediate values ​​and subranges therebetween.

[0116] In some of the embodiments described herein, the method further includes exposing the hardened modeling material formulation to post-processing conditions before or after removal of the support material formulation, if the support material formulation is included in the build material. The post-processing conditions are typically intended to further solidify the hardened modeling material. In some embodiments, the partially cured formulation is solidified by post-processing to obtain a fully cured formulation.

[0117] In some embodiments, the post-treatment is by exposure to heat or radiation, as described in any of the embodiments herein.

[0118] In some embodiments, it is contemplated that different formulations may be dispensed from different dispensing heads to manufacture objects, in particular providing the ability to select a formulation from a predefined number of formulations and define the selected formulation and a desired combination of its properties.

[0119] According to this embodiment, the spatial location of deposition of each formulation in the layer is determined such that different formulations occupy different three-dimensional spatial locations, or such that two or more different formulations occupy substantially the same three-dimensional location or adjacent three-dimensional locations, allowing spatial combination of the formulations within the layer after deposition.

[0120] Thus, the present embodiment is capable of depositing a wide range of material combinations and producing objects comprised of different combinations of multiple modeling material formulations, where the multiple modeling material formulations can be located in different portions of the object according to the properties desired to characterize each portion of the object.

[0121] Systems utilized in additive manufacturing may include a receiving medium and one or more dispensing heads. The receiving medium may be, for example, a manufacturing tray that may have a horizontal surface that carries the material dispensed from the print head. In some embodiments, the receiving medium is formed or coated with a biocompatible material, as described herein.

[0122] The dispensing head can be, for example, a print head having a plurality of dispensing nozzles arranged in one or more arrays along a longitudinal axis of the dispensing head, and the dispensing head can be positioned such that its longitudinal axis is substantially parallel to the indexing direction.

[0123] The additive manufacturing system may further include a controller, such as a microprocessor, that controls the AM process, e.g., the operation of the dispensing head according to a predetermined scan plan (e.g., a CAD configuration converted into Standard Tessellation Language (STL) format and programmed into the controller). The dispensing head may include multiple jetting nozzles. The jetting nozzles dispense material onto a receiving medium to form layers that represent a cross-section of the 3D object.

[0124] In addition to the dispensing head, a source of curing energy can be provided for curing the dispensed build material. The curing energy is typically radiation, e.g., ultraviolet or thermal radiation. Alternatively, means can be provided for providing curing conditions other than electromagnetic or thermal radiation, such as means for cooling the dispensed build material or means for contacting the build material with a reagent that promotes curing.

[0125] Additionally, the AM system may include a leveling device to level and / or define the height of each layer after deposition and at least partial solidification and before depositing the next layer.

[0126] According to the present embodiment, the additive manufacturing methods described herein are for bioprinting biological objects.

[0127] As used herein, "bioprinting" refers to performing an additive manufacturing process utilizing one or more bio-ink formulations that include biological components as described herein in a manner compatible with an automated or semi-automated computer-aided additive manufacturing system (e.g., a bioprinter or bioprinting system) as described herein.

[0128] Throughout this specification, the phrase "modeling material formulation" (also referred to herein interchangeably as "modeling formulation" or "modeling material composition" or "modeling composition" or simply "formulation" or "composition") refers to some or all of the uncured build material (print medium) that is dispensed to form a final object, as described herein. A modeling formulation is an uncured modeling formulation that, upon exposure to curing conditions, forms an object or part thereof.

[0129] In the context of bioprinting, the uncured build material includes at least one modeling formulation that includes one or more biological components or materials (e.g., the curable rh collagen described herein), also referred to herein and in the art as a "bio-ink" or "bio-ink formulation."

[0130] In some embodiments, bioprinting involves the sequential formation of multiple layers of uncured build material in a construction pattern, preferably according to three-dimensional printing data as described herein. At least one, and preferably most or all, of the formed layers comprises (before solidifying or curing) one or more biological components as described herein (e.g., curable rh collagen as described herein). Optionally, at least one of the formed layers comprises (before solidifying or curing) one or more non-biological curable materials, and / or non-curable biological or non-biological components, preferably biocompatible materials that do not interfere with (e.g., do not adversely affect) the biological and / or structural characteristics of the biological components (e.g., collagen) in the print medium and / or bioink.

[0131] In some embodiments, the components in the bio-ink or printed medium, e.g., the non-curable and curable materials, and / or the curing conditions applied to effect curing, are selected so as not to significantly affect the structural and / or functional properties of the biological components in the bio-ink or printed medium.

[0132] In some of the embodiments described herein, the build material (e.g., print medium) comprises a modeling material formulation (bio-ink) and optionally a support material formulation, all selected to comprise a material or combination of materials that do not interfere with the biological and / or structural characteristics of the biological components.

[0133] In some of the embodiments described herein, the bioprinting methods are configured to form layers under conditions that do not significantly affect the structural and / or functional properties of the biological components in the bio-ink.

[0134] In some embodiments, a bioprinting system for carrying out the bioprinting processes / methods described herein is configured to allow layers to be formed under conditions that do not significantly affect the structural and / or functional properties of the biological components in the bio-ink.

[0135] In some of the embodiments described herein, additive manufacturing (e.g., bioprinting) processes and systems are configured such that process parameters (e.g., temperature, shear force, shear strain rate) do not interfere with (substantially affect) the functional and / or structural characteristics of the biological components.

[0136] In some of the embodiments described herein, the additive manufacturing process (bioprinting) is carried out at a temperature of at least 10°C or at least 20°C, for example, at a temperature in the range of about 10 to about 40°C, preferably about 10°C to 37°C, or about 20°C to 37°C, or about 20°C to about 30°C, or about 20°C to about 28°C, or about 20°C to about 25°C (including intermediate values ​​and subranges therebetween), or at room temperature, or at 37°C.

[0137] In some of the embodiments described herein, the temperatures / temperature ranges mentioned above are the temperatures at which the build material (e.g., the modeling material formulation including at least a biological component as described herein) is dispensed, i.e., the temperature of the dispensing head in the AM system and / or the temperature at which the modeling material formulation is held before passing through the dispensing head.

[0138] In some of the embodiments described herein, the AM process is performed without cooling the AM system (e.g., the dispensing head and / or the modeling material formulation) to a temperature below room temperature, e.g., below 20°C or below 10°C, or below 5°C (e.g., 4°C).

[0139] In some of the embodiments described herein, the AM system does not have a means to cool the system or parts thereof (e.g., the dispensing head and / or the modeling material formulation) to a temperature below room temperature, e.g., below 20°C or below 10°C, or below 5°C (e.g., 4°C).

[0140] In some of the embodiments described herein, additive manufacturing processes (bioprinting) are performed while applying shear forces that do not adversely affect the structural and / or functional properties of the biological components (e.g., cells). The application of shear forces can be performed by passing a build material (e.g., a modeling material formulation including at least the biological components described herein) through a dispensing head, which should also be considered as subjecting the build material to shear forces.

[0141] As described herein and shown in the Examples section below, embodiments of the invention allow the AM bioprinting process to be carried out under conditions (e.g., low shear and room or physiological temperatures) that do not affect the functional and / or structural characteristics of the biological components contained in the bioink, while maintaining the necessary fluidity (viscosity that confers fluidity, e.g., less than 10,000 centipoise, or less than 5,000 centipoise, or less than 2,000 centipoise) and further maintaining the hardenability of the dispensed build material. Embodiments of the invention are well suited to operating bioprinting using any of the known methods and are not limited to the process parameters required for each such method.

[0142] Below, examples of AM bioprinting methods that can be used in embodiments of the present invention are described.

[0143] The bioprinting method and corresponding system can be any of the methods and systems known in the art for additive manufacturing, examples of such systems and methods are described above. Suitable methods and systems can be selected taking into account printing capabilities such as resolution, deposition rate, scalability, bioink compatibility, and ease of use.

[0144] For example, a suitable bioprinting system typically includes a dispensing system (either with a temperature control module or at room temperature), a stage (receiving medium), and motion along the x, y, and z axes as directed by CAD-CAM software. A curing source (e.g., light or heat source) and / or humidifier that applies curing energy (e.g., applying light or thermal radiation) or curing conditions to the deposition area (receiving medium) to promote hardening of the formed layer may also be included in the system. Printers exist that use multiple dispensing heads to facilitate sequential dispensing of several materials.

[0145] In general, bioprinting can be performed using known techniques for additive manufacturing. Some exemplary additive manufacturing techniques are listed below, although any other techniques are contemplated.

[0146] 3D Inkjet Printing: 3D inkjet printing is a common 3D printer used for both non-biological and biological (bioprinting) applications. Inkjet printers use thermal or acoustic forces to jet droplets onto a substrate that can support or form part of the final structure. This technique delivers a controlled amount of liquid to a predefined location, resulting in high-resolution prints with precise control over (1) the location of the ink droplets and (2) the amount of ink (which is beneficial for printing microstructures or when adding small amounts of bioreactive reagents or drugs). Inkjet printers can be used with several inks, including, for example, multiple biological components and / or bioactive agents. Furthermore, printing is fast and can be applied to culture plates.

[0147] A bioprinting method utilizing a 3D inkjet printing system can be performed using one or more bio-ink modeling material formulations described herein, dispensing droplets of the formulation in layers onto a receiving medium using one or more inkjet printheads according to the 3D printing data.

[0148] Extrusion Printing: This technique uses continuous beads of material instead of droplets. These beads of material are deposited in 2D, with the stage (receiving medium) or extrusion head moving along the z-axis, with the deposited layer serving as the foundation for the next layer. The most common methods of biological material extrusion for 3D bioprinting applications are pneumatic or mechanical dispensing systems.

[0149] Stereolithography (SLA) and Digital Light Processing (DLP): SLA and DLP are additive manufacturing techniques that convert uncured build material in a bath into solidified material, layer by layer, by selective curing using a light source, while later separating / washing the uncured material from the solidified material. SLA is widely used in a variety of industries, including bioprinting, to create models, prototypes, patterns, and production parts.

[0150] Laser printing: A form of laser-based printing technology adapted for 3D bioprinting is based on the principle of Laser-Induced Forward Transfer (LIFT), which was developed to transfer metals and has now been successfully applied to biological materials. The device consists of a laser beam, a focusing system, an energy absorption / transduction layer, a layer of biological material (e.g. cells and / or hydrogels) and a receiving substrate. Laser-based printers work by irradiating the absorption layer with a laser beam, which converts the energy into a mechanical force that extrudes small droplets from the biological layer onto the substrate. A light source is then used to harden the material on the substrate.

[0151] Laser printing is compatible with a range of viscosities and allows printing of mammalian cells without affecting cell viability or cell function. Cell deposition can be up to 10 8 It can be performed at a density of 1 cell / ml and with microscale resolution of one cell per drop.

[0152] Electrospinning: Electrospinning is a fiber-producing technique that uses electrical forces to draw electrically charged threads of a polymer solution or melt.

[0153] Recombinant Human Collagen: As used herein, the term "collagen" refers to a polypeptide having a triple helical structure and containing repeating Gly-XY triplets, where X and Y can be any amino acid, but are often imino acids (proline and hydroxyproline). According to one embodiment, the collagen is type I, II, III, V, XI, or biologically active fragments derived therefrom.

[0154] Collagen of the present invention also refers to homologs (e.g., polypeptides that are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95% or more, about 100% homologous to the collagen sequences set forth in Table A as determined by the BlastP software of the National Center for Biotechnology Information (NCBI) using default parameters). Homologs may also refer to deletion, insertion or substitution variants thereof (including amino acid substitutions) and biologically active polypeptide fragments thereof.

[0155] According to a particular embodiment, the collagen is human collagen.

[0156] In another embodiment, the collagen comprises the naturally occurring amino acid sequence of human collagen.

[0157] Table A below provides examples of collagen NCBI SEQ ID NOs.

[0158] [Table 1]

[0159] The annotation for SEQ ID NO:1 is as follows: Amino acids 1-22: signal peptide Amino acids 23-161: N-terminal peptide Amino acids 162 to 1218: collagen α-1(I) chain Amino acids 1219-1464: C-terminal peptide

[0160] The annotation for SEQ ID NO:2 is as follows: Amino acids 1-22: signal peptide Amino acids 23-79: N-terminal peptide Amino acids 80 to 1119: collagen α-2(I) chain Amino acids 1120 to 1366: C-terminal peptide

[0161] According to one embodiment, the collagen of the present invention contains sufficient telopeptides to enable it to form fibrils under appropriate conditions.

[0162] Thus, for example, the collagen can be atelocollagen, telocollagen or procollagen.

[0163] As used herein, the term "atelocollagen" refers to a collagen molecule that lacks both the N-terminal and C-terminal propeptides normally contained in procollagen and at least some of its telopeptides, but contains enough of the telopeptides such that it is capable of forming fibrils under appropriate conditions.

[0164] As used herein, the term "procollagen" refers to a collagen molecule (e.g., human) that includes either an N-terminal propeptide or a C-terminal propeptide, or both. Exemplary human procollagen amino acid sequences are shown in SEQ ID NOs: 3, 4, 5, and 6.

[0165] As used herein, the term "telocollagen" refers to a collagen molecule that lacks both the N- and C-terminal propeptides normally contained in procollagen, but contains telopeptides. The telopeptides of fibrillar collagen are remnants of the N- and C-terminal propeptides following digestion with native N / C proteinases.

[0166] According to other embodiments, the collagen lacks its telopeptides and is unable to undergo fibrillogenesis.

[0167] According to another embodiment, the collagen is a mixture of the collagen types mentioned above.

[0168] According to certain embodiments, the collagen is genetically engineered by recombinant DNA techniques (eg, human collagen).

[0169] Methods for isolating collagen from animals are known in the art. Dispersion and solubilization of native animal collagen can be accomplished using a variety of proteolytic enzymes (e.g., porcine mucosal pepsin, bromelain, chymopapain, chymotrypsin, collagenase, ficin, papain, peptidase, proteinase A, proteinase K, trypsin, microbial proteases, and similar enzymes or combinations of such enzymes) that disrupt intermolecular bonds and remove immunogenic non-helical telopeptides without affecting the essential rigid triple helical structure that confers collagen's desirable properties (see U.S. Pat. Nos. 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037; 3,949,073; 4,233,360; and 4,488,911 for general methods of preparing purified soluble collagen). The resulting soluble collagen can then be purified by repeated precipitation at low pH and high ionic strength, followed by washing and resolubilization at low pH.

[0170] Plants expressing collagen chains and procollagens are known in the art (see, e.g., WO06035442A3; Merle et al., FEBS Lett. 2002 Mar 27;515(1-3):114-8. PMID: 11943205; and Ruggiero et al., 2000, FEBS Lett. 2000 Mar 3;469(1):132-6. PMID: 10708770; and U.S. Patent Application Publication Nos. 2002 / 098578 and 2002 / 0142391; and U.S. Patent No. 6,617,431), each of which is incorporated herein by reference.

[0171] It will be understood that the present invention also contemplates genetically modified collagens / atelocollagens, such as collagenase-resistant collagens, etc. [Wu et al., Proc Natl. Acad Sci, Vol. 87, p.5888-5892, 1990].

[0172] Recombinant pro- or telocollagen (eg, human) can be expressed in any non-animal cell, including, but not limited to, plant cells and other eukaryotic cells such as yeast and fungi.

[0173] Plants capable of producing (i.e., expressing) procollagen or telocollagen can be lower (e.g., mosses and algae) or higher (vascular) plant species (e.g., tissues or isolated cells and extracts thereof (e.g., cell suspensions)). Preferred plants are those that are 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 resistance to stress conditions and the ease with which the expressed components or aggregated 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.

[0174] Recombinant procollagen is usually produced by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen.

[0175] Exemplary polynucleotide sequences encoding human procollagen are shown in SEQ ID NOs:7, 8, 9 and 10.

[0176] Human telocollagen is usually produced 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, or the protease can be added after isolation of the recombinant procollagen.

[0177] The stability of the triple helical structure of collagen requires the hydroxylation of proline by the enzyme prolyl-4-hydroxylase (P4H) to form hydroxyproline residues within the collagen chain. Although plants can synthesize hydroxyproline-containing proteins, the prolyl hydroxylases involved in the synthesis of hydroxyproline in plant cells exhibit relatively loose substrate sequence specificity compared to mammalian P4H. Thus, the production of collagen containing hydroxyproline only at the Y position of the Gly-XY triplet requires the co-expression of collagen and human or mammalian P4H genes [Olsen et al, Adv Drug Deliv Rev. 2003 Nov 28;55(12):1547-67].

[0178] Thus, according to one embodiment, procollagen or telocollagen is expressed in an intracellular compartment of a plant that does not have endogenous P4H activity.

[0179] As used herein, the phrase "subcellular compartment with no 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. According to one embodiment, the subcellular compartment is a vacuole, apoplast, or chloroplast. According to a particular embodiment, the subcellular compartment is a vacuole. According to another embodiment, the subcellular compartment is an apoplast.

[0180] Accumulation of expressed procollagen in intracellular compartments that lack endogenous P4H activity can be achieved by any one of several approaches.

[0181] For example, the expressed procollagen / telocollagen can contain a signal sequence for targeting the expressed protein to a subcellular compartment, such as the apoplast or an organelle (eg, chloroplast).

[0182] Examples of suitable signal sequences include chloroplast transit peptide (contained in SwissProt entry P07689, amino acids 1-57) and mitochondrial transit peptide (contained in SwissProt entry P46643, amino acids 1-28). Targeting to the vacuole can be achieved by fusing the collagen-encoding polynucleotide sequence to a vacuolar targeting sequence, for example using the vacuolar targeting sequence of the thiol protease alurein precursor (NCBI accession number P05167 GI:113603): MAHARVLLLALAVLATAAVAVASSSSFADSNPIRPVTDRAASTLA (SEQ ID NO: 14). Typically, the collagen-encoding polynucleotide sequence also includes an ER targeting sequence. In one embodiment, the ER targeting sequence is native to the collagen sequence. In other embodiments, the native ER targeting sequence is removed and a non-native ER targeting sequence is added. The non-native ER targeting sequence may be included in the vacuolar targeting sequence. It will be understood that to translocate across the ER to the vacuole, the collagen sequence should not include an ER retention sequence.

[0183] Alternatively, the sequence of the procollagen can be modified in a way that alters the cellular localization of the procollagen when expressed in plants.

[0184] The present invention contemplates genetically modified cells that co-express both human procollagen and P4H. In one embodiment, P4H can precisely hydroxylate the procollagen α-chain (i.e., hydroxylate 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 numbers P07237 and P13674. Both subunits are necessary to form the active enzyme, but the β subunit also has a chaperone function.

[0185] The P4H expressed by the genetically modified cells of the present invention is preferably human P4H. Exemplary polynucleotide sequences encoding human P4H are SEQ ID NOs: 11 and 12. In addition, P4H mutants exhibiting high substrate specificity, or P4H homologs, can also be used. An example of a suitable P4H homolog includes the Arabidopsis oxidoreductase identified under NCBI Accession No. NP_179363.

[0186] Since it is essential that P4H co-accumulates with the expressed procollagen chain, it is preferable to appropriately modify its coding sequence (e.g., by adding or deleting a signal sequence). Thus, the present invention contemplates the use of P4H polynucleotide sequences fused to a vacuolar targeting sequence. It will be appreciated that in order to target the vacuole, if an endogenous ER retention sequence is present, it must be removed prior to expression.

[0187] In mammalian cells, collagen is also modified by lysyl hydroxylases, galactosyltransferases, and glucosyltransferases. 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 sequential modification steps seen in the formation of hydroxylysine-linked carbohydrates.

[0188] Thus, the genetically modified cells of the invention can also express mammalian LH3 (optionally fused to a vacuolar targeting sequence), it being understood that in order to target to the vacuole, the endogenous ER retention sequence is removed prior to expression.

[0189] An LH3 coding sequence such as that shown in SEQ ID NO:13 may be used for such purposes.

[0190] The procollagens and modifying enzymes described above can be expressed from stably integrated or transiently expressed nucleic acid constructs that contain a polynucleotide sequence encoding a procollagen alpha chain and / or a modifying enzyme (e.g., P4H and LH3) under the transcriptional control of a functional promoter. Such nucleic acid constructs (also referred to herein as expression constructs) can be configured for expression in the entire organism (e.g., in a plant, in a defined tissue or cell) and / or at a defined developmental stage of the organism. Such constructs can also include selectable markers (e.g., antibiotic resistance), enhancer elements, and origins of replication for bacterial replication.

[0191] A variety of methods exist 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 on stable integration of the nucleic acid construct, or a portion thereof, into the plant genome or on transient expression of the nucleic acid construct, where such sequences are not inherited by the plant's progeny.

[0192] Additionally, several methods exist by which nucleic acid constructs can be directly introduced into the DNA of DNA-containing organelles such as chloroplasts.

[0193] There are two principle methods for achieving stable genomic integration of exogenous sequences, such as those contained within the nucleic acid constructs of the present invention, into a plant genome.

[0194] (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, CJ, Butterworth Publishers, Boston, Mass. (1989) p. 93-112. (ii) Direct uptake of DNA: 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) p. 52-68; Toriyama, K. et al. (1988) Bio / Technology 6:1072-1074, including a method for direct uptake of DNA into protoplasts. Uptake of DNA induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or plant tissues by particle bombardment, Klein et al. Bio / Technology (1988) 6:559-563; McCabe et al. al. Bio / Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by 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 germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, GP and Mantell, SH and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

[0195] There are various methods for direct DNA introduction 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.

[0196] Regardless of the transformation technique used, once collagen-expressing progeny are identified, such plants are further grown under conditions that maximize their expression. Progeny resulting from transformed plants can be selected by confirming the presence of exogenous mRNA and / or polypeptide using nucleic acid or protein probes (e.g., antibodies). The latter approach also validates the plant's potential for correct processing and assembly of foreign proteins, since the expressed polypeptide components can be localized (e.g., by probing fractionated plant extracts).

[0197] After cultivation of such plants, the telopeptide-containing collagen is usually harvested. The plant tissue / cells are harvested, preferably at maturity, and the procollagen molecules are isolated by an extraction approach. The harvesting is preferably carried out such that the procollagen remains in a state that can be cleaved by protease enzymes. According to one embodiment, a crude extract is produced from the transgenic plant of the invention and then contacted with the protease enzyme.

[0198] As described above, the propeptide- or telopeptide-containing collagen can be solubilized after incubation with a protease to produce atelocollagen or collagen. It will be appreciated that the propeptide- or telopeptide-containing collagen can be purified from the genetically modified cells before incubation with the protease or after incubation with the protease. Alternatively, the propeptide- or telopeptide-containing collagen can be partially purified before protease treatment and completely purified after protease treatment. Alternatively, other extraction / purification procedures can be performed simultaneously with the treatment of the propeptide- or telopeptide-containing collagen with a protease.

[0199] Examples of methods for purifying or semi-purifying the telopeptide-containing collagen of the present invention include, but are not limited to, salting out with ammonium sulfate or the like and / or removing small molecules by ultrafiltration.

[0200] According to one embodiment, the proteases used to cleave the recombinant propeptide- or telopeptide-containing collagen are not of animal origin. Exemplary proteases include, but are not limited to, certain plant-derived proteases, such as ficin (EC 3.4.22.3), and certain bacterial-derived proteases, such as subtilisin (EC 3.4.21.62) and neutrase. The inventors also contemplate the use of recombinant enzymes such as rhtrypsin and rhpepsin. Several such enzymes are commercially available, such as ficin from fig tree latex (Sigma, Cat. No. F4125 and Europe Biochem), subtilisin from Bacillus licheniformis (Sigma, Cat. No. P5459), neutrase from Bacillus amyloliquefaciens (Novozymes, Cat. No. PW201041) and TrypZean™, recombinant human trypsin expressed in maize (Sigma, Cat. No. T3449).

[0201] In some of the embodiments described herein, the recombinant human collagen is recombinant human type I collagen.

[0202] In some of the embodiments described herein, the recombinant human collagen is plant-derived recombinant human collagen, and in some embodiments, the plant is tobacco.

[0203] In some of the embodiments described herein, the recombinant human collagen is a recombinant human type I collagen comprising two α1 units having an amino acid sequence that is at least 90% identical, at least 91% identical, 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical to the sequence set forth in SEQ ID NO:15 as determined by the BlastP software of the National Center for Biotechnology Information (NCBI) using default parameters, and one α2 unit having an amino acid sequence that is at least 90% identical, at least 91% identical, 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical to the sequence set forth in SEQ ID NO:6. According to a particular embodiment, the type I collagen consists of two α1 units consisting of the sequence set forth in SEQ ID NO: 15 and one α2 unit consisting of the sequence set forth in SEQ ID NO: 6 as determined by the BlastP software of the National Center for Biotechnology Information (NCBI) using default parameters.

[0204] In some of the embodiments described herein, the α1 unit is encoded by a polynucleotide sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100%, identical to the nucleic acid sequence set forth in SEQ ID NO:16. The α2 unit is encoded by a polynucleotide sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100%, identical to the nucleic acid sequence set forth in SEQ ID NO:10.

[0205] Identity (eg, percent homology) can be determined using any homology comparison software, such as the BlastN software of the National Center for Biotechnology Information (NCBI) using default parameters, etc.

[0206] In some of the embodiments described herein, the human recombinant collagen (rhcollagen) described in any of the embodiments herein is monomeric rhcollagen.

[0207] By "monomeric" is meant rh collagen as described herein that is soluble in aqueous solution and does not form fibrillar aggregates.

[0208] In some of the embodiments described herein, the human recombinant collagen (rh collagen) described in any of the embodiments herein is fibrillar rh collagen.

[0209] By "fibrous" is meant rh collagen as described herein that takes the form of fibrillar aggregates in aqueous solution. Typically, but not necessarily, fibrillar rh collagen is formed by exposing monomeric rh collagen to a fibrillogenesis buffer, typically characterized by a basic pH. An exemplary procedure for forming fibrillar rh collagen is described in the Examples section below.

[0210] Sclerosing collagen: According to one aspect of some embodiments of the present invention, a composition is provided that includes a hardenable collagen. According to some embodiments, the composition can be used in additive manufacturing (e.g., bioprinting) of 3D objects as described herein. According to some embodiments, the composition can be used in or is for preparing a modeling material formulation for additive manufacturing processes (e.g., bioprinting).

[0211] "Curable" as used herein means a material that is capable of hardening or solidifying, as defined herein, upon exposure to appropriate curing conditions.

[0212] Curable materials typically harden or harden through polymerization and / or crosslinking.

[0213] A hardenable material is typically a polymerizable material that polymerizes and / or crosslinks when exposed to appropriate curing conditions or appropriate curing energy (appropriate energy source). Alternatively, the hardenable material is a thermoresponsive material that hardens or solidifies when exposed to a temperature change (e.g., heating or cooling). In some cases, the hardenable material is made of small particles (e.g., nanoparticles or nanoclays) that can harden to form a solidified material. In still other cases, the hardenable material is a biological material that undergoes a biological reaction (e.g., an enzyme-catalyzed reaction) to form a hardened or solid material.

[0214] In some of the embodiments described herein, the curable material is a photopolymerizable material that polymerizes and / or crosslinks upon exposure to radiation as described herein, hi some embodiments, the curable material is a UV curable material that polymerizes or crosslinks upon exposure to UV-Visible light as described herein.

[0215] In some of the embodiments described herein, when the curable material is exposed to curing conditions (e.g., radiation, reagents), it polymerizes by any or a combination of chain extension, entanglement, and crosslinking. Crosslinking can be chemical and / or physical.

[0216] In some of the embodiments described herein, the curable material can be a monofunctional curable material or a multifunctional curable material.

[0217] As used herein, a monofunctional curable material contains one curable group, i.e., a functional group capable of polymerizing, entangling, and / or crosslinking upon exposure to curing conditions (e.g., radiation, presence of calcium ions).

[0218] A multifunctional curable material contains two or more, e.g., two, three, four or more, curable groups. A multifunctional curable material can be, for example, a difunctional, trifunctional or tetrafunctional curable material containing two, three or four curable groups, respectively.

[0219] "Sclerotic collagen" refers to collagen as described herein in the embodiments (e.g., human recombinant collagen) having one or more sclerotic groups as defined herein. According to some of the embodiments described herein, the sclerotic collagen is a multifunctional sclerotic material that contains multiple sclerotic groups as defined herein.

[0220] The terms "sclerotic collagen," "sclerotic rh collagen," and "rh collagen having one or more (or at least one) sclerotic group" are used interchangeably herein.

[0221] According to some of the embodiments described herein, the hardenable collagen comprises an amino acid sequence as described herein in each embodiment and has one or more (preferably a plurality) hardenable groups formed by covalent attachment of a compound containing a hardenable group to functional groups, preferably in the side chains of at least some of the amino acid residues forming the collagen. Alternatively or additionally, the hardenable group can be formed at the N-terminus and / or C-terminus of one or more units forming the collagen, for example by covalent attachment of a compound containing a hardenable group to an amine or carboxylic acid.

[0222] According to some of the embodiments described herein, at least a portion of the hardenable groups of the hardenable collagen described herein are crosslinkable groups and undergo crosslinking upon exposure to curing conditions.

[0223] In some embodiments, the curable groups are capable of polymerizing and / or crosslinking via a free radical mechanism.

[0224] Examples of such curable groups include acrylic groups, such as acrylic acid groups, methacrylic acid groups, acrylamide groups, and methacrylamide groups. Other free radical curable groups can include thiols, vinyl ethers, and other groups characterized by a reactive double bond.

[0225] In some embodiments, the curable groups can be polymerized and / or crosslinked by other mechanisms, such as cationic polymerization or (cationic or anionic) ring-opening polymerization. Examples of such curable groups include, but are not limited to, epoxy-containing groups, caprolactam, caprolactone, oxetane, and vinyl ethers.

[0226] Other curable groups include, for example, the formation of an amide bond between a functional carboxylic acid and an amine group (each being a curable group that reacts with the other to crosslink), the formation of a urethane between an isocyanate group and a hydroxyl group by polycondensation in the presence of a catalyst and / or upon exposure to ultraviolet light, and the formation of a disulfide bond between two thiols.

[0227] Any other hardenable groups are contemplated.

[0228] The generation of hardenable groups on the hardenable collagen can be accomplished by direct chemical reaction of a material that contains or is capable of generating hardenable groups with a chemically compatible functional group present on the collagen, as described herein, or by means of a spacer or linker using chemistries well known in the art. For example, a material that contains hardenable groups and functional groups can be reacted with a compatible functional group on the collagen (e.g., a functional group on an amino acid side chain) such that the hardenable group becomes a substituent on the amino acid side chain.

[0229] In some embodiments, the compatible functional groups are first generated within the collagen by chemical modification of the collagen's chemical groups, which react with a material that contains or generates hardenable groups upon reaction.

[0230] When the hardenable collagen contains more than one hardenable group, the hardenable groups may be the same or different.

[0231] According to some of the embodiments described herein, at least some or all of the hardenable groups in the hardenable collagen of the present embodiments are photopolymerizable groups (e.g., ultraviolet light-curable groups) that can polymerize and / or crosslink upon exposure to radiation as described herein.

[0232] According to some of the embodiments described herein, the hardenable group is a photohardenable group or a photopolymerizable group (eg, an acrylate or a methacrylate).

[0233] Alternatively or additionally, the curable groups are thiol-containing groups, which upon curing result in disulfide cross-links.

[0234] Alternatively or additionally, the hardenable groups or moieties are hardened by conjugation (using a coupling agent such as EDC) or by a chemical reaction such as glycation.

[0235] According to some embodiments, the hardenable groups include amine and carboxyl groups that form peptide bonds upon curing.

[0236] According to some of the embodiments described herein, at least some or all of the hardenable groups in the hardenable collagen of the present embodiments are acrylic groups as defined herein.

[0237] According to some of the embodiments described herein, acrylic groups such as methacrylamide can be generated by reacting an acrylate or methacrylate (e.g., acrylic acid, methacrylic acid, acrylic or methacrylic esters, acrylic or methacrylic anhydrides) with an amine functional group (e.g., of a lysine residue).

[0238] According to some embodiments of the present invention, the degree of hardening (e.g., degree of cross-linking) can be determined by the number of hardenable groups in the hardenable collagen described herein, and the number can be manipulated to obtain a desired degree of hardening (e.g., degree of cross-linking).

[0239] According to some of the embodiments described herein, the hardenable collagen has multiple acrylamide or methacrylamide hardenable groups generated by reaction with lysine residues as described herein.

[0240] According to some of the embodiments described herein, the hardenable collagen has a plurality of acrylamide or methacrylamide hardenable groups that replace the amine groups of lysine residues in the collagen.

[0241] In some embodiments, at least 50%, or at least 60%, or at least 70% of the lysine residues in the collagen are substituted with methacrylamide or acrylamide groups. In some embodiments, the hardenable collagen is characterized in that 70%-100%, or 80%-100%, or 90%-100% of its lysine residues (including any intermediate values ​​and subranges therebetween) are substituted with methacrylamide or acrylamide groups.

[0242] According to one aspect of some embodiments of the present invention, there is provided a process for preparing the curable rh collagen described herein, which in some embodiments is carried out by reacting a material that includes or generates curable groups with rh collagen in accordance with embodiments described herein.

[0243] The number of hardenable groups in the rh collagen can be controlled by manipulating the amount of material that reacts with the rh collagen to generate hardenable groups.

[0244] In some embodiments, materials are used in molar excess relative to each functional group that reacts with the material, e.g., chemically compatible functional groups in rh collagen (e.g., amino acid residues characterized by such functional groups) or rh collagen, e.g., in a molar ratio of 2:1, 5:1, 10:1, 15:1, 20:1, 30:1, 50:1, or, for example, 1.1:1 to 50:1 (including intermediate values ​​and subranges therebetween).

[0245] According to some of the embodiments described herein, the sclerotic collagen is recombinant human type I collagen as described herein in any of the embodiments and any combination thereof.

[0246] Modeling Material Formulation: According to the present embodiments described herein, in at least one of the layers formed during the AM (bioprinting process), the dispensed modeling material formulation comprises a biological component or material as described herein and is considered a bio-ink as described herein.

[0247] According to this embodiment, the build material comprises at least one modeling material formulation comprising a curable recombinant human collagen, as described in any of the embodiments herein, such modeling material formulations are also referred to herein as rh collagen-containing formulations.

[0248] According to some of the embodiments described herein, the rh collagen-containing formulation further comprises a carrier, and in some of these embodiments, the carrier is an aqueous carrier.

[0249] The aqueous carrier can be water, a buffer solution characterized by a pH in the range of about 4 to about 10, or about 6 to about 8, or about 7 to about 7.4, a basic aqueous solution, or an acidic aqueous solution.

[0250] Aqueous carriers can contain salts and other water-soluble materials at various concentrations, hi some embodiments, the concentration of salt in the carrier is in the range of about 0.1 mM to about 0.2 M, or about 0.1 mM to about 0.1 M, or about 0.1 mM to about 100 mM, or about 0.1 mM to about 50 mM, or about 0.1 mM to about 20 mM, including any intermediate or subrange therebetween.

[0251] In some embodiments, the aqueous carrier contains a physiologically acceptable concentration of salt such that the formulation is characterized by an osmolality near physiological osmolality.

[0252] In some embodiments, the aqueous carrier comprises a phosphate, such as monobasic sodium phosphate (NaH2PO4) and / or dibasic sodium phosphate (sodium hydrogen phosphate, Na2HPO4). In some embodiments, the total concentration of the phosphate is about 0.1 M.

[0253] In some embodiments, the aqueous carrier comprises NaCl or other physiologically acceptable salt.

[0254] In some embodiments, the aqueous carrier comprises a phosphate buffer, and in some embodiments, the aqueous carrier comprises a phosphate buffered saline solution comprising sodium phosphate monobasic and / or sodium phosphate dibasic and NaCl.

[0255] Phosphate buffered saline (PBS) can be commercially available PBS (eg, DPBS) or a custom buffer characterized by the desired pH and / or osmolality.

[0256] In an exemplary embodiment, the aqueous carrier comprises a phosphate buffer comprising a sodium phosphate salt described herein at a concentration of about 0.1 M and NaCl at a concentration of about 0 mM to about 200 mM, including any intermediate values ​​and subranges therebetween.

[0257] Any other buffer may be used in the context of this embodiment.

[0258] In some of the embodiments described herein, the aqueous carrier comprises an acid.

[0259] In some embodiments, the concentration of the acid is less than 100 mM, for example, 0.1 mM to 50 mM, or 0.1 mM to 30 mM, or 0.1 mM to 40 mM, or 0.1 mM to 30 mM, or 1 to 30 mM, or 10 to 30 mM (including any intermediate values ​​or subranges therebetween).

[0260] The acid can be an inorganic acid (eg, HCl) or an organic acid (preferably water soluble at the concentrations described above (eg, acetic acid)).

[0261] In some of the embodiments described herein, the aqueous carrier comprises a culture medium. The culture medium can be a commercially available culture medium or a custom culture medium. The culture medium can be any liquid medium that at least allows cell survival. Such media can include, for example, salts, sugars, amino acids, and minerals at appropriate concentrations along with various additives, and one of skill in the art can determine the appropriate medium for a particular cell type. Non-limiting examples of such media include phosphate buffered saline, DMEM, MEM, RPMI 1640, McCoy's 5A medium, Medium 199, and IMDM (available, for example, from Biological Industries, Beth Haemek, Israel, Gibco-Invitrogen Corporation, Grand Island, NY, USA).

[0262] The medium may be supplemented with various antibiotics (eg, penicillin and streptomycin), growth factors or hormones, specific amino acids (eg, L-glutamine), cytokines, and the like.

[0263] In some of the embodiments described herein, the concentration of the curable recombinant human collagen in the modeling material formulation is in the range of 0.5 mg / mL to 50 mg / mL, or 0.5 mg / mL to 20 mg / mL, or 1 mg / mL to 50 mg / mL, or 1 mg / mL to 50 mg / mL, or 1 mg / mL to 40 mg / mL, or 1 mg / mL to 30 mg / mL, or 2 mg / mL to 20 mg / mL, or 5 mg / mL to 15 mg / mL (including any intermediate values ​​or subranges therebetween).

[0264] The concentration of curable rh collagen in the modeling material formulation can affect the rheological properties of the formulation and the resulting solidified formulation upon dispensing, and can be manipulated depending on the AM method and conditions used and the desired properties of the final object or portion thereof.

[0265] According to some of the embodiments described herein, the modeling material formulation is characterized by shear thinning behavior and is a shear thinning formulation.

[0266] According to some of the embodiments described herein, the modeling material formulation is characterized by thermal thinning behavior and a thermal thinning formulation.

[0267] The term "shear thinning" describes a property of a fluid material that is reflected in a decrease in viscosity (increased fluidity) upon the application of shear force (under shear strain). In some embodiments, a shear thinning material exhibits a significant decrease in its shear modulus (e.g., at least 100%) as the shear strain is increased from about 1% to greater than 50%. Thus, a shear thinning material exhibits a shear force dependent viscosity profile.

[0268] The term "thermally thinning" describes the property of a fluid material reflected in a decrease in viscosity (increased fluidity) upon the application of thermal energy (increased temperature). In some of the present embodiments, a thermally thinning material is characterized by a decrease in viscosity of at least 10% upon a temperature increase of 5° C. Thus, a thermally thinning material exhibits a temperature dependent viscosity profile.

[0269] According to some of the embodiments described herein, the rh collagen-containing formulation is both a shear-thinning formulation and a thermally thinning formulation, as defined herein.

[0270] According to some of the embodiments described herein, the modeling material formulation is characterized by a viscosity of 2,000 centipoise or less, or 1,500 centipoise or less, at zero shear rate and 37° C., as measured using a rheometer as described in the Examples section below.

[0271] According to some of the embodiments described herein, the modeling material formulation is characterized by a viscosity of 2,000 centipoise or less, or 1,500 centipoise or less, at a shear rate of 5 1 / sec and room temperature, as measured using a rheometer as described in the Examples section below.

[0272] According to some of the embodiments described herein, the modeling material formulation is characterized by a viscosity as described above when the concentration of hardenable collagen is at least 3 mg / mL, or at least 5 mg / mL, e.g., 10 mg / mL.

[0273] According to some of the embodiments described herein, the modeling material formulation is characterized by a viscosity profile at 37°C that is similar to the viscosity profile at 4°C.

[0274] According to some of the embodiments described herein, the modeling material formulation is characterized by a viscosity at 37°C that is at least 10%, or at least 20%, lower than the viscosity at 4°C measured at shear rates of 1-10 l / sec for the same concentration of curable rh collagen.

[0275] According to some of the embodiments described herein, the modeling material formulation is characterized by a storage modulus (G') of at least 1,000 Pa upon solidification.

[0276] According to some of the embodiments described herein, the modeling material formulation is characterized by an increase in its storage modulus (G') upon solidification (e.g., upon exposure to the curing conditions described in any of the embodiments herein) of at least 10 times.

[0277] According to some of the embodiments described herein, the modeling material formulation, upon solidification, forms a hydrogel material by crosslinking of the rh collagen within the aqueous carrier.

[0278] As used herein and in the art, the term "hydrogel" refers to a three-dimensional fibrous network that contains at least 20%, usually at least 50%, or at least 80%, and up to about 99.99% (by mass) of water. Hydrogels can be considered as materials that are mostly water but behave like a solid or semi-solid due to a three-dimensional cross-linked solid-like network formed by polymer chains (e.g., collagen chains) in a liquid dispersion medium. The polymer chains are interconnected (cross-linked) by chemical bonds (covalent, hydrogen, and ionic / complex / metallic bonds, usually covalent).

[0279] Throughout this specification, when polymer chains or polymeric materials are mentioned, this includes polymeric biological materials (eg, macromolecules) such as peptides, proteins, oligonucleotides, and nucleic acids.

[0280] Hydrogels can take physical forms ranging from soft, brittle and weak to hard, elastic and tough. Soft hydrogels are characterized by rheological parameters including elastic and viscoelastic parameters, while stiff hydrogels are properly characterized by tensile strength parameters, elastic modulus, storage modulus and loss modulus, as these terms are known in the art.

[0281] The softness / hardness of a hydrogel is governed, inter alia, by the chemical composition of the polymer chains, the "degree of cross-linking" (the number of interconnecting links between the chains), the content and composition of the aqueous medium, and the temperature.

[0282] According to some embodiments of the present invention, the hydrogel may include polymeric polymeric and / or fibrous elements that are not chemically bound to the main crosslinked network, but rather undergo mechanical entanglement and / or immersion. Such polymeric fibrous elements may be woven (e.g., as in a mesh structure) or nonwoven, and in some embodiments may function as a reinforcing material for the hydrogel's fibrous network. Non-limiting examples of such polymers include polycaprolactone, gelatin, gelatin methacrylate, alginate, alginate methacrylate, chitosan, chitosan methacrylate, glycol chitosan, glycol chitosan methacrylate, hyaluronic acid (HA), HA methacrylate, and other non-crosslinked natural or synthetic polymer chains. Alternatively or additionally, such polymers may be chemically bound to the main crosslinked network of the hydrogel, for example, by acting as a crosslinker or forming part of the three-dimensional network of the hydrogel.

[0283] In some embodiments, the hydrogel is porous, and in some embodiments, at least a portion of the pores in the hydrogel are nanopores, having an average volume in the nanoscale range.

[0284] According to some of the embodiments described herein, the rh collagen-containing modeling material formulation further comprises one or more additional materials, such as one or more additional hardenable materials, one or more non-hardenable materials, and / or one or more biological components or materials.

[0285] According to some of the embodiments described herein, the print medium (build material) includes one or more additional materials, such as one or more additional hardenable materials, one or more non-hardenable materials, and / or one or more biological components.

[0286] According to some of the embodiments described herein, additional materials are included in the rh collagen-containing formulation or one or more other modeling material formulations.

[0287] Additional hardenable materials that may be included in the rh collagen formulation or one or more other modeling material formulations can be any hardenable material as defined herein, and are preferably biocompatible materials.

[0288] In some embodiments, the further hardenable material is or comprises a hydrogel, as defined herein, and upon exposure to curing conditions in which crosslinking and / or copolymerization reactions occur, typically by further crosslinking and / or copolymerization, can form a solidified modeling material. Such hardenable materials are also referred to herein as hydrogel hardenable materials.

[0289] In some of the embodiments described herein, the curable material is or comprises a hydrogel-forming material, as defined herein, that is capable of forming a hydrogel as a solidified modeling material upon exposure to curing conditions that cause crosslinking, polymerization, and / or copolymerization, and / or entanglement reactions, typically by crosslinking, entanglement, polymerization, and / or copolymerization. Such curable materials are also referred to herein as hydrogel-forming curable materials or gel-forming materials.

[0290] According to embodiments of the present invention, the hydrogel may be of biological origin or synthetically prepared.

[0291] According to some embodiments of the invention, the hydrogel is biocompatible and the activity of the biological moiety is maintained when the biological moiety is impregnated or accumulated in the hydrogel, i.e. the activity of the biological moiety changes by no more than 30%, or no more than 20%, or no more than 10% compared to the activity of the biological moiety in a physiological medium.

[0292] Examples of polymers or copolymers that can be used to form hydrogels according to the present invention include polyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides, polyvinylpyrrolidones, and copolymers of any of the above. Other examples include polyethers, polyurethanes, and poly(ethylene glycols), which can be functionalized with crosslinking groups or used in combination with compatible crosslinkers.

[0293] Some specific, non-limiting examples include poly(2-vinylpyridine), poly(acrylic acid), poly(methacrylic acid), poly(N-isopropylacrylamide), poly(N,N'-methylenebisacrylamide), poly(N-(N-propyl)acrylamide), poly(methacrylic acid), poly(2-hydroxyacrylamide), poly(ethylene glycol) acrylate, poly(ethylene glycol) methacrylate, and polysaccharides such as hyaluronic acid, dextran, alginate, agarose, and copolymers of any of the above.

[0294] Hydrogel precursors (hydrogel-forming materials) that form such polymer chains (including any combination thereof) are contemplated.

[0295] Hydrogels are typically formed from or in the presence of di-, tri- or polyfunctional monomers, oligomers or polymers, collectively referred to as hydrogel precursors or hydrogel formers or materials having two, three or more polymerizable groups. The presence of more than one polymerizable group makes such precursors crosslinkable, allowing for the formation of a three-dimensional network.

[0296] Examples of crosslinkable monomers include, but are not limited to, the family of diacrylate and triacrylate monomers having two or three polymerizable functional groups, one of which can be considered the crosslinkable functional group. Examples of diacrylate monomers include methylene diacrylate, and poly(ethylene glycol). nExamples of triacrylate monomers include, but are not limited to, trimethylolpropane triacrylate, pentaerythritol triacrylate, tris(2-hydroxyethyl)isocyanurate triacrylate, isocyanuric acid tris(2-acryloyloxyethyl)ester, ethoxylated trimethylolpropane triacrylate, pentaerythrityl triacrylate and glycerol triacrylate, phosphinylidintris(oxyethylene)triacrylate.

[0297] In some of the embodiments described herein, the curable material, whether monomeric or oligomeric, can be a monofunctional curable material or a multifunctional curable material.

[0298] Examples of polymers or copolymers that can be used to form hydrogels according to the present invention include polyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides, polyvinylpyrrolidones, and copolymers of any of the above. Other examples include polyethers, polyurethanes, and poly(ethylene glycols), which can be functionalized with crosslinking groups or used in combination with compatible crosslinkers.

[0299] Some specific, non-limiting examples include poly(2-vinylpyridine), poly(acrylic acid), poly(methacrylic acid), poly(N-isopropylacrylamide), poly(N,N'-methylenebisacrylamide), poly(N-(N-propyl)acrylamide), poly(methacyclic acid), poly(2-hydroxyacrylamide), poly(ethylene glycol) acrylate, poly(ethylene glycol) methacrylate, and polysaccharides such as dextran, alginate, agarose, and copolymers of any of the above.

[0300] Hydrogel precursors (hydrogel-forming materials) that form such polymer chains (including any combination thereof) are contemplated.

[0301] Curable materials that can be used in the field of bioprinting are mainly based on naturally occurring materials that can be isolated from animal or human tissues, such as Matrigel, alginate, pectin, xanthan gum, gelatin, chitosan, fibrin, cellulose and hyaluronic acid, or recombinantly produced or synthetically prepared materials, such as polyethylene glycol; PEG, gelatin methacrylate; GelMA, poly(propylene oxide); PPO, poly(ethylene oxide); PEO; PEG, polyethylene glycol. poly(dimethylsiloxane); nanocellulose; Fmoc-peptide based hydrogels such as Pluronic F127, short dipeptides (FF), Fmoc-FF-OH, Fmoc-FRGD-OH, Fmoc-RGDF-OH, Fmoc-2-Nal-OH, Fmoc-FG-OH, and thermoplastic polymers such as polycaprolactone (PCL), polylactic acid (PLA) or poly(D,L-lactide-co-glycolide).

[0302] Examples of curable materials that can be used in the context of the present embodiments include, but are not limited to, Matrigel, gelatin methacrylate (GelMA), nanocellulose (UV-curable nanoscale structured materials such as cellulose nanocrystals (CNC), cellulose nanofibrils (CNF), and bacterial cellulose (BC), also referred to as microbial cellulose), Pluronic® materials such as Pluronic F127 and UV-curable Pluronic F127-diacrylate (DA), which are flowable at low temperatures and form gels at elevated temperatures above the critical micelle concentration (CMC), hyaluronic acid (HA), acrylated hyaluronic acid (AHA), methacrylated hyaluronic acid (MAHA), poly(ethylene glycol) diacrylate (PEGDA), alginates, xanthan gum, pectin, glutaraldehyde, chitosan that can be crosslinked with chemical agents such as genipin or sodium tripolyphosphate (TPP).

[0303] Exemplary curable materials are described in the Examples section below.

[0304] Biological components or materials that can be included in one or more of the modeling material formulations described herein include, for example, cellular components such as cultured cells and other cellular components such as cytokines, chemokines, growth factors, and other biological components such as proteins, agents that act to enhance cell adhesion, cell spreading, cell proliferation, cell differentiation, and / or cell migration, amino acids, peptides, polypeptides, proteins, DNA, RNA, lipids, and / or proteoglycans.

[0305] The cells may comprise a heterogeneous cell population, or the cells may comprise a homogeneous cell population. Such cells may be, for example, stem cells (such as embryonic stem cells, bone marrow stem cells, umbilical cord blood cells, mesenchymal stem cells, adult tissue stem cells, etc.), progenitor cells, or differentiated cells, such as chondrocytes, osteoblasts, connective tissue cells (such as fibrocytes, fibroblasts, and adipocytes), endothelial cells, and epithelial cells. The cells may be untreated or genetically modified.

[0306] According to one embodiment of this aspect of the invention, the cells are of mammalian origin.

[0307] Additionally, the cells may be of autologous or non-autologous origin, such as postpartum-derived cells (as described in U.S. Patent Application Nos. 10 / 887,012 and 10 / 887,446). Generally, the choice of cells will depend on the desired use.

[0308] Suitable proteins that can be used include extracellular matrix proteins [e.g., fibrinogen, collagen, fibronectin, vimentin, microtubule-associated protein 1D, neurite outgrowth factor (NOF), bacterial cellulose (BC), laminin, and gelatin], cell adhesion proteins [e.g., integrins, proteoglycans, glycosaminoglycans, laminins, intercellular adhesion molecule (ICAM) 1, N-CAM, cadherins, tenascin, gicerin, RGD peptide, and nerve injury-induced protein 2 (ninjurin 2)], growth factors [epidermal growth factor, transforming growth factor-α, fibroblast growth factor-acidic, bone morphogenetic proteins, fibroblast growth factor-basic, erythropoietin, thrombopoietin, hepatocyte growth factor, insulin-like growth factor-I, insulin-like growth factor-II, interferon-β, platelet-derived growth factor, vascular endothelial growth factor and angiopeptin], cytokines [e.g., M-CSF, IL-1β, IL-8, β-thromboglobulin, EMAP-II, G-CSF and IL-10], proteases [pepsin, low specificity chymotrypsin, high specificity chymotrypsin, trypsin, carboxypeptidases, aminopeptidases, proline-endopeptidases, Staphylococcus aureus V8 protease, proteinase K (PK), aspartic proteases, serine proteases, metalloproteases, ADAMTS17, tryptase-γ and matriptase-2] and protease substrates.

[0309] Additionally, calcium phosphate materials such as hydroxyapatite can be used, for example in the form of particles (including but not limited to nano-HA and nano-TCP). The particle size must be compatible with the dispensing head to avoid clogging.

[0310] The non-hardenable materials other than the biological materials described herein that may be included in one or more of the modeling formulations described herein may be materials that impart certain properties to the formulation or to the solidified formulation and part of the object formed therefrom. Such properties may be physical properties (e.g., optical properties such as transparency or opacity, color, spectral properties, heat resistance, electrical properties, etc.), or mechanical or rheological properties such as viscosity, elasticity, storage modulus, loss modulus, stiffness, hardness, etc. Alternatively or additionally, the non-hardenable material may provide a biological function, for example, a therapeutically active agent.

[0311] Examples of non-curable materials include thixotropic agents, reinforcing agents, toughening agents, fillers, colorants, pigments, and the like.

[0312] An example of a non-curable material is titanium dioxide.

[0313] An example of a non-hardening material is oxidized cellulose.

[0314] According to some of the embodiments described herein, one or more of the modeling material formulations includes hyaluronic acid.

[0315] According to some of the embodiments described herein, one or more of the modeling material formulations includes hyaluronic acid having hardenable groups, as defined herein.

[0316] According to some of the embodiments described herein, one or more of the modeling material formulations include one or more biological components or materials (e.g., but not limited to, cells, growth factors, peptides, heparan sulfate, and fibronectin).

[0317] According to some of the embodiments described herein, one or more of the modeling material formulations include one or more agents that modify the mechanical properties of the compositions and / or objects described herein, including, but not limited to, alginate, hyaluronic acid, fibrinogen, elastin, peptides, and thixotropic agents (e.g., crystalline nanocellulose (CNC)), oxidized cellulose, titanium dioxide, clay minerals, and carbon nanotubes.

[0318] In some of the embodiments described herein, the rh collagen-containing formulation further comprises one or more additional hardenable materials as described in any of the embodiments herein.

[0319] In some of these embodiments, the weight ratio of hardenable recombinant human collagen to additional hardenable material is in the range of 10:1 to 1:2, or 10:1 to 1:1, or 5:1 to 2:1 (including any intermediate or subrange therebetween).

[0320] In some of the embodiments described herein, the rh collagen-containing formulation further comprises a thixotropic agent as defined herein.

[0321] Throughout this specification, the term "thixotropy" refers to the property of a fluid compound or material that is reflected in a time-dependent shear thinning, i.e., its viscosity decreases as a function of the duration of the application of a shear force, and returns to its original value when the application of the shear force is discontinued. In some of the present embodiments, the thixotropic material or agent exhibits a significant (e.g., at least 100%) decrease in shear modulus under 50% strain.

[0322] In some of the embodiments described herein, the rh collagen-containing formulation further comprises a gel forming agent, such as a hydrogel forming agent described herein.

[0323] In some of the embodiments described herein, the rh collagen-containing formulation further comprises a biological component or material as described herein.

[0324] In some of the embodiments described herein, the rh collagen-containing formulation further comprises one or more hardenable or non-hardenable materials as described in any of the embodiments herein.

[0325] According to some of the embodiments described herein, the recombinant human collagen is selected from monomeric collagen and fibrillar collagen, as demonstrated in the Examples section below.

[0326] According to some of the embodiments described herein, the molar ratio of curable groups to collagen is essentially as set forth in the Examples section herein.

[0327] According to some of the embodiments described herein, the formulation further comprises hyaluronic acid as described herein.

[0328] According to some of the embodiments described herein, the formulation further comprises hyaluronic acid characterized by a polymerizable group.

[0329] According to some of the embodiments described herein, the formulation further comprises one or more biological components, such as, but not limited to, components selected from cells, growth factors, peptides, heparan sulfate, and fibronectin.

[0330] According to some of the embodiments described herein, the formulation further comprises one or more agents that modify the mechanical properties of the formulation and / or object, including, but not limited to, alginate, hyaluronic acid, fibrinogen, elastin, peptides, and thixotropic agents (e.g., crystalline nanocellulose (CNC)).

[0331] According to some of the embodiments described herein, the formulations are characterized by a neutral pH (eg, about 6 to about 8).

[0332] According to some of the embodiments described herein, the formulation is essentially characterized by the viscosity parameters described herein.

[0333] According to some of the embodiments described herein, the formulation is characterized by having approximately the same viscosity at temperatures above 4° C. and 10° C., or above 20° C. (e.g., 37° C.).

[0334] In embodiments using two or more modeling material formulations, the two or more formulations are rh collagen-containing formulations as described herein that differ from each other in the presence, type and / or concentration of additional materials included therein. For example, one formulation can include curable rh collagen and another formulation can include curable rh collagen and a biological material as described herein. For example, one formulation can include curable rh collagen and another formulation can include curable rh collagen and an additional curable material as described herein. For example, one formulation can include curable rh collagen and an additional curable material and another formulation can include curable rh collagen and another additional curable material as described herein. For example, one formulation can include curable rh collagen and an additional curable material and another formulation can include curable rh collagen and a non-curable material as described herein. Any other combinations are contemplated.

[0335] In some of the embodiments described herein, all of the curable materials in the build material are cured under the same curing conditions, hi some embodiments, all of the curable materials are photocurable.

[0336] In some of the embodiments described herein, the modeling material formulations that include a hardenable material further include an agent that promotes the hardening or solidification of the hardenable material upon exposure to curing conditions.

[0337] The concentration of the reagents can be determined depending on the concentration of the curable material and the desired degree of cure (eg, the desired degree of crosslinking).

[0338] When the curable material is a photocurable material, the agent is a photoinitiator. The photoinitiator is selected depending on the curing mechanism (e.g., free radical, cationic, etc.).

[0339] A free radical photoinitiator can be any compound that generates free radicals when exposed to radiation, such as ultraviolet or visible light, thereby initiating a polymerization reaction. Non-limiting examples of suitable photoinitiators include benzophenones (aromatic ketones), such as benzophenone, methylbenzophenone, Michler's ketone, and xanthone; acylphosphine oxide type photoinitiators, such as 2,4,6-trimethylbenzolydiphenylphosphine oxide (TMPO), 2,4,6-trimethylbenzoylethoxyphenylphosphine oxide (TEPO), and bisacylphosphine oxide (BAPO); benzoins and benzoin alkyl ethers, such as benzoin, benzoin methyl ether, and benzoin isopropyl ether. Examples of photoinitiators include α-aminoketones and bisacylphosphine oxides (BAPO).

[0340] Examples of photoinitiators include, but are not limited to, the Irgacure® family, riboflavin, rose bengal, and the like.

[0341] Free radical photoinitiators may be used alone or in combination with coinitiators. Coinitiators are used with initiators that require a second molecule to generate active radicals in photocurable free radical systems. Benzophenone is an example of a photoinitiator that requires a second molecule, such as an amine, to generate free radicals. After absorbing radiation, benzophenone reacts with tertiary amines by hydrogen abstraction to generate α-amino radicals that initiate the polymerization of acrylates. Non-limiting examples of types of coinitiators include alkanolamines such as triethylamine, methyldiethanolamine, and triethanolamine.

[0342] Suitable cationic photoinitiators include, for example, compounds that form aprotic or Bronsted acids upon exposure to sufficient ultraviolet and / or visible light to initiate polymerization. The photoinitiator used can be a single compound, a mixture of two or more active compounds, or a combination of two or more different compounds, i.e., coinitiators. Non-limiting examples of suitable cationic photoinitiators include aryl diazonium salts, diaryliodonium salts, triarylsulfonium salts, triarylselenonium salts, and the like. One example of a cationic photoinitiator is a mixture of triarylsulfonium hexafluoroantimonate salts.

[0343] Non-limiting examples of suitable cationic photoinitiators include P-(octyloxyphenyl)phenyliodonium hexafluoroantimonate UVACURE 1600 (available from Cytec Company, USA), iodonium (4-methylphenyl)(4-(2-methylpropyl)phenyl)-hexafluorophosphate known as Irgacure 250 or Irgacure 270 (available from Ciba Specialty Chemicals, Switzerland), mixed arylsulfonium hexafluoroantimonate salts known as UVI 6976 and 6992 (available from Lambson Fine Chemicals, UK), diaryliodonium hexafluoroantimonate known as PC 2506 (available from Polyset Company, USA), (tolylcumyl)iodonium tetrakis(pentafluorophenyl)borate known as Rhodorsil® Photoinitiator 2074 (available from Bluestar Silicones, USA), Tego and iodonium bis(4-dodecylphenyl)-(OC-6-11)-hexafluoroantimonate, known as PC1466 (available from Evonik Industries AG, Germany).

[0344] object: Throughout this specification, in the context of bioprinting, the term "object" refers to the end product of additive manufacturing that includes at least a biological component in its entirety. This term refers to the product obtained after removal of a support material, if one is used as part of the uncured build material in the bioprinting methods described herein.

[0345] As used throughout this specification, the term "object" refers to the entire object or a portion thereof.

[0346] In the context of this embodiment, the object comprises at least a portion of a collagen-based material.

[0347] By "collagen-based material" is meant a material that comprises collagen, preferably a material that comprises recombinant human collagen as described herein in any of the embodiments and any combination thereof.

[0348] In some of the embodiments described herein, the collagen-based material comprises a scaffold, for example a hydrogel scaffold formed of a three-dimensional fibrous network comprising recombinant human collagen as described herein.

[0349] In some of the embodiments described herein, the collagen-based material comprises polymerized and / or cross-linked recombinant human collagen in which multiple monomeric and / or fibrillar collagen units are linked together to form a three-dimensional network.

[0350] The three-dimensional network or scaffold can be, for example, a film, a sponge, a porous structure, a hydrogel, and any other form depending on the desired need.

[0351] In some of the embodiments described herein, the object is in the form of a tissue or organ and comprises at least a portion of the collagen-based material described herein. Such objects can be assembled according to the respective 3D printing data of the desired organ or tissue using the hardenable collagen described herein as well as additional hardenable and biological materials described herein.

[0352] In some embodiments, the object is an implantable object. In some embodiments, the object is artificial skin. In some embodiments, the object is artificial tissue (e.g., connective tissue or muscle tissue, such as cardiac tissue or pancreatic tissue). Examples of connective tissue include, but are not limited to, cartilage (such as elastic cartilage, hyaline cartilage, and fibrocartilage), adipose tissue, reticular connective tissue, embryonic connective tissue (such as mesenchymal connective tissue and mucous connective tissue), tendon, ligament, and bone.

[0353] In some embodiments, the object is usable or intended for construction of a prosthetic organ or tissue.

[0354] The object may further comprise a solidified material formed from one or more of the additional hardenable materials described in any of the embodiments herein, biological components or materials described in any of the embodiments herein, and / or non-hardenable materials described in any of the embodiments herein.

[0355] In some embodiments, the article is in the form of a collagen scaffold or film that can be used for research or therapeutic applications, such as repair of damaged tissue (eg, when seeding damaged tissue with cultured cells) or wound healing.

[0356] The scaffolds can be administered to a subject in need thereof to regenerate tissues such as connective tissue, muscle tissues such as cardiac tissue and pancreatic tissue.

[0357] The films can be used to construct biomedical devices, such as collagen membranes for hemodialysis.

[0358] According to some embodiments, the film or scaffold may be used in cell culture.

[0359] As used herein, the phrase "cell culture" or "culture" refers to the maintenance of cells in an artificial, e.g., in vitro environment. However, it is to be understood that the term "cell culture" is a general term and can be used to include not only the culture of individual prokaryotic (e.g., bacterial) or eukaryotic (e.g., animal, plant and fungal) cells, but also the culture of tissues, organs, organ systems or whole organisms.

[0360] In some embodiments, the films or scaffolds may be used in the wound healing process.

[0361] In some embodiments, the collagen films described herein can be used to prevent adhesions after tendon injury, to extend ophthalmic surgery of the levator palpebrae superioris, to repair severed nerves, etc. Additionally, the collagen films described herein can be used in burn dressings and for healing bone defects.

[0362] The purposes of this embodiment include countless other uses, including, but not limited to, the treatment of diseases such as interstitial cystitis, scleroderma, rheumatoid arthritis, cosmetic surgery, as an aid in the healing of burn patients, as a wound healing agent, as a dermal filler, for spinal fusion procedures, for urethral dilation, in duraplasty procedures, for bone reconstruction, and for various dental, orthopedic and surgical purposes.

[0363] kit: According to an aspect of some embodiments of the present invention there is provided a kit comprising a sclerotic rh collagen as described herein in any of the embodiments.

[0364] According to some embodiments, the kit comprises a composition comprising the sclerotic rh collagen described in any of the embodiments herein.

[0365] According to some embodiments, the composition comprises curable rh collagen in lyophilized form.

[0366] According to some embodiments, the composition comprises a curable rh collagen and an aqueous solution or aqueous carrier.

[0367] According to some of the embodiments described herein, the kit is specified or usable for use as a modeling material formulation for additive manufacturing (e.g., bioprinting) of an object described in any of the embodiments herein.

[0368] According to some of the embodiments described herein, the kit further comprises an aqueous carrier, as described in any of the embodiments herein, hi some embodiments, the composition and the aqueous carrier are packaged separately within the kit.

[0369] Alternatively, the kit includes instructions for mixing the composition with an aqueous carrier to prepare a modeling material formulation as described herein.

[0370] It is expected that many related curable biocompatible materials, bioprinting media, and / or bioprinting techniques will be developed during the life of the patent that matures from this application, and the scope of the embodiments relating to curable biocompatible materials, bioprinting media, and / or bioprinting techniques is intended to encompass all such new technologies a priori.

[0371] As used herein, the term "about" means ±10% or ±5%.

[0372] The terms "comprises," "comprising," "includes," "including," "having" and conjugations thereof mean "including, but not limited to."

[0373] The term "consisting of" means "including and limited to."

[0374] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0375] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" includes a plurality of compounds, and may include mixtures thereof.

[0376] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and is not an inflexible limitation of the scope of the invention. Thus, the description of a range should be considered to specifically disclose all the possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose not only the subranges 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., but also each individual numerical value within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the magnitude of the range.

[0377] When a numerical range is given herein, it is intended to include any recited number (fractional or integer) within the range given. The phrases "range between" a first designated number and a second designated number and "range from" a first designated number to a second designated number are used interchangeably herein and are intended to include the first designated number and the second designated number, and all fractional and integer numbers therebetween.

[0378] As used herein, the term "method" means manner, means, techniques, and procedures for accomplishing a given task, including, but not limited to, those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine, or those that can be readily developed by practitioners from known manners, means, techniques, and procedures.

[0379] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating the clinical or cosmetic symptoms of a condition, or substantially preventing the appearance of clinical or cosmetic symptoms of a condition.

[0380] It will be understood that features of the invention that are described for clarity in the context of separate embodiments may also be provided in a single embodiment in any combination of those features. Conversely, features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination, as appropriate, in other described embodiments of the invention. Features described in the context of various embodiments should not be construed as essential features of that embodiment unless the embodiment is inoperative without that element.

[0381] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0382] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting manner.

[0383] material: Human recombinant collagen (rhcollagen) type I, expressed and isolated from transgenic tobacco plants, was produced and supplied by CollPlant Ltd (Israel).

[0384] Type I bovine collagen (PureCol®) and bovine methacrylated collagen were purchased from Advanced Biomatrix (USA).

[0385] Methacrylic anhydride, glycidyl methacrylate, triethylamine, tetrabutylammonium bromide, 2,4,6-trinitrobenzenesulfonic acid (TNBS), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), sodium phosphate monobasic anhydrous, HCl 1N, HCl ≥ 37%, sodium bicarbonate, titanium dioxide (TiO2), Hepes buffer, eosin Y, N-vinylpyrrolidone, and NaOH were purchased from Sigma Aldrich Ltd (Israel).

[0386] Dulbecco's phosphate buffered saline (DPBS), ×10 DPBS, fetal bovine serum (FBS), DMEM high glucose and penicillin / streptomycin were purchased from Biological Industries Ltd (Israel).

[0387] Sodium phosphate dibasic anhydrous was purchased from Canton (India).

[0388] Absolute ethanol and acetone were purchased from Bio-Lab Ltd (Israel).

[0389] Hyaluronic acid was purchased from Lifecore (USA).

[0390] Acetic acid was purchased from JT Baker or Advanced Biometrix.

[0391] Oxidized cellulose was received from Synthesia.

[0392] PVAMA (polyvinyl alcohol methacrylate) was kindly provided by Professor Shoseyov.

[0393] TEA (triethanolamine) was purchased from JT Baker.

[0394] Nano-hydroxyapatite (nano-HA) and nano-tricalcium phosphate (nano-TCP) were purchased from Fluidinova.

[0395] Preparation of buffer stock solutions: Fibril Formation Buffer (FB): Dibasic sodium phosphate was dissolved in double distilled water (DDW) to a final concentration of 162 mM. The solution was adjusted to pH 11.2 with 10 N NaOH.

[0396] Preparation of medium: 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.

[0397] Preparation of phosphate buffer (0.1M) / saline (0.016M) (300mOs / L): 1 M phosphate buffer was prepared as follows: 31.6 mL of 1 M sodium phosphate dibasic was mixed with 68.4 mL of 1 M sodium phosphate monobasic. The resulting 1 M phosphate buffer was diluted to 0.1 M by adding DDW (9:1, vol / vol). NaCl was added to a final concentration of 15.8 mM to obtain a physiological osmolality (300 mOs / L).

[0398] Preparation of phosphate buffer (0.1 M; pH 7.4) / saline (11.3 mM) (physiological osmolarity): 1 M phosphate buffer was prepared as follows: 77.4 mL of 1 M sodium phosphate dibasic was mixed with 22.6 mL of 1 M sodium phosphate monobasic. The resulting 1 M phosphate buffer was diluted to 0.1 M by adding DDW (9:1, vol / vol). NaCl was added to a final concentration of 11.3 mM to obtain a physiological osmolality (300 mOs / L).

[0399] Washing Buffer: HCl was added to the fibrillogenesis buffer to a final concentration of 16.2 mM dibasic sodium phosphate and 10 mM HCl. The pH was adjusted to 7.2-7.4 with 10 N NaOH.

[0400] Equipment data: 1H-NMR spectra were recorded at 25 °C on a Bruker Avance II spectrometer (1H resonance frequency: 500 MHz, solvent used: D2O). Analysis was performed by quantitative NMR using 64 scans for averaging signal-to-noise ratio and a recycle delay of 8.6 seconds. The values ​​obtained are expressed in ppm (standard chemical shift) δ.

[0401] Viscosity and G' / G" measurements were performed on a HAAKE RheoStress600 rheometer (Thermo Electron Corporation) equipped with a temperature-controlled cell chamber using a C60 / 1°Ti or C35 / 1°Ti cone-plate setup. Analysis of the crosslinked (solidified) disks was performed using a PP20 sawtooth spindle and 20 mm sawtooth plate setup.

[0402] In situ crosslinking experiments were carried out by a TA Discovery Hybrid temperature-controlled rheometer equipped with a UV chamber.

[0403] Other measurements were performed as described below.

[0404] [Example 1] Methacrylation of rh collagen Fibrillar rh collagen-methacrylamide and monomeric rh collagen-methacrylamide were prepared by reacting lysine and hydroxylysine residues of collagen with methacrylic anhydride in an aqueous medium as described below, and stored at 4° C., protected from light, until further use.

[0405] Fibrillar rh collagen-methacrylamide: Fibrillar rhcollagen-methacrylamide (rhcollagen-MA) was synthesized at 3-10 mg / mL in 1x wash buffer described herein, fibrillogenesis buffer described herein, or DDW at room temperature (RT) or 12 °C.

[0406] In an exemplary procedure, fibrous rhcollagen-MA was synthesized in DDW as follows: 3-4 mg / mL of monomeric rhcollagen in 10 mM HCl solution (Collage™) was mixed with fibril formation buffer in a 9:1 v / v ratio and stirred at room temperature for 1 h to obtain fibrils. This solution was centrifuged at 7500 rpm and 4°C for 30 min, after which the supernatant was discarded. The remaining pellet was resuspended in the same amount of washing buffer and centrifuged under the same conditions. The precipitated fibrils were then resuspended in DDW to obtain a 10 mg / mL solution. The concentration was confirmed by % solids measurement. Methacrylic anhydride was added dropwise at room temperature in a 10-20 molar ratio (10-20 times) to collagen lysine under a nitrogen flow, and the pH of the reaction solution was monitored over time and adjusted to pH 7 with 10 N NaOH. After 24 h of reaction, the mixture was dialyzed against wash buffer (pH = 7) using 100 kDa cut-off dialysis tubing (Spectrum Laboratories Inc, California, USA) at 4 °C for 3 days, and the dialysate (wash buffer in this case) was exchanged at least six times to remove reaction by-products, followed by lyophilization for 3–4 days.

[0407] The degree of modification of rh collagen was quantified by a TNBS (2,4,6-trinitrobenzenesulfonic acid) colorimetric assay, which quantifies the molar content of free unreacted ε-amino groups derived from lysine and hydroxyl-lysine and the corresponding degree of functionalization.

[0408] The assay was performed according to a previously described protocol [Sashidhar et al., Journal of Immunological Methods. 1994, 167, 121-127] and based on Habeeb [Habeeb AFSA, Analytical Biochemistry. 1966, 14, 328-336]. Briefly, 0.4 mL of freshly prepared 0.01% (v / v) TNBS was added to 0.4 mL of 0.1-2 mg / mL fibrillar rhcollagen-MA 4% sodium bicarbonate solution. After 2 h of reaction at 40°C, 0.2 mL of 1N HCl and 0.4 mL of 10% (v / v) SDS were added. Absorbance measurements were performed at 335 nm in a 1 mL polystyrene cuvette in a spectrophotometer. A control (blank) was prepared following the same procedure, except that sodium bicarbonate buffer was added instead of the rhcollagen-MA solution. The absorption of 1-2 mg / mL native fibrous rh collagen prepared under the same conditions was recorded for calibration.

[0409] Table 1 below shows data obtained on the degree of functionalization (measured by TNBS assay) of batches obtained by reacting fibrous rh collagen with 10x, 15x or 20x molar ratios of methacrylic anhydride.

[0410] [Table 2]

[0411] These results indicate that all MA concentrations result in a high degree of modification of fibrillar rh collagen, suggesting that the addition of methacrylic reagent at a molar ratio of 10:1 is preferred for maximal functionalization of fibrillar rh collagen.

[0412] Monomeric rh collagen-methacrylamide: Synthesis of monomeric rhcollagen-methacrylamide was carried out using 200 mM MOPS, phosphate or Tris buffers supplemented with 150 mM NaCl.

[0413] In an exemplary procedure, 200 mM MOPS and 150 mM NaCl were added to 3-4 mg / mL collage™ (monomeric rh collagen in 10 mM HCl), and the solution was stirred at room temperature until a clear solution was obtained. A 1-20 fold molar excess of methacrylic anhydride was then added dropwise at 12°C under a nitrogen stream, and the pH was adjusted to 7 over time with 10 N NaOH. After 24 h of reaction, the mixture was first dialyzed against 10 mM HCl / 20 mM NaCl (pH=2) using a 100 kDa cutoff dialysis hollow fiber cartridge (GE Healthcare), with at least five changes of dialysate, and then dialyzed against 10 mM HCl. The material was then freeze-dried.

[0414] The structural characteristics of unmodified monomeric rhcollagen and the extent of methacrylation of collagen lysine amines (also referred to herein as Coll-MA) of rhcollagen were investigated. 1 The collagen was analyzed by H-NMR spectroscopy (500 MHz). A representative spectrum of Coll-MA synthesized with a 10-fold molar excess of methacrylic anhydride is shown in Figure 1. The signals of the methyl functional group and the acrylic protons of the introduced methacrylate are shown in (b) and (c), respectively, and the lysine methylene signals of unmodified rh collagen are shown in (a).

[0415] The spectrum of unmodified rh collagen was in high agreement with the collagen spectrum reported in the literature [R. Ravichandran et.al; J. Mater. Chem. B, 2016, 4, 318-326].

[0416] The degree of methacrylation can be calculated from the spectra obtained using quantitative analysis techniques. The signal at δ = 3 ppm (a) is due to the lysine methylene groups of collagen and was significantly reduced after methacrylation. In comparison with unmodified rh-collagen, new signals at 1.9 ppm (b) and 5.4-5.7 ppm (c) (attributed to the methyl functional group and acrylic protons of the introduced methacrylic group, respectively) were observed in the spectrum of Coll-MA, confirming the successful conjugation of methacrylate to collagen.

[0417] [Example 2] Viscosity measurements of rh-collagen and rh-collagen-MA Preparation of solutions for viscosity measurements: PureCol® and Collage™ in DPBS: 8 mL of monomeric collagen solution (3 mg / mL in 10 mM HCl) with rh collagen (Collage™) or bovine collagen (PureCol™) was neutralized by adding 1 mL of DPBS×10. The solution was then adjusted to pH 7-7.5 with 0.1 N NaOH. Double distilled water (DDW) was then added to a final volume of 10 mL. Samples were incubated at 37°C for at least 90 min before measurements were taken (at 37°C or 4°C).

[0418] 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 (at 37° C. or 4° C.).

[0419] Fibrillar rh collagen-methacrylamide in DPBS: Lyophilized fibrillar rhcollagen-MA, prepared in DDW and dialyzed against wash buffer (described in Example 1 above, using a 10:1 molar ratio of MA:rhcollagen), was dissolved in DPBS to a concentration of 10 mg / mL. Samples were incubated at 37° C. for at least 90 minutes prior to measurements (at 37° C. or 4° C.).

[0420] Monomeric rh collagen-methacrylamide in acetic acid: A 10 mg / ml solution of rh collagen-methacrylamide in 20 mM acetic acid was prepared by adding the appropriate amount of medium to the lyophilized material and mixing at room temperature until completely dissolved.

[0421] Bovine collagen-methacrylate in acetic acid: A 10 mg / ml solution of bovine collagen-methacrylate in 20 mM acetic acid was prepared by adding the appropriate amount of vehicle (Advanced BioMatrix) to the lyophilized material and mixing at room temperature until completely dissolved.

[0422] Temperature dependence of rh collagen / bovine collagen viscosity: Figure 2 shows comparative plots of the viscosity of rh collagen (Collage™) and bovine collagen (PureCol) in DPBS as a function of shear rate at T = 4°C (blue, dashed and solid lines, respectively) and T = 37°C (red, dashed and solid lines, respectively). Bovine collagen (solid line) shows a clear temperature dependence of the zero shear rate viscosity (η0) (i.e., a viscosity plateau at low shear rate values), with the η0 value at 37°C (red) being more than an order of magnitude higher than that at 4°C (blue). In contrast, rh collagen (dashed line) shows no significant difference between the η0 values ​​at 4°C and 37°C.

[0423] FIG. 3 is a comparative plot showing the viscosity of rh collagen neutralized with FB, prepared as described above, which shows very similar behavior (i.e., the viscosity at 4° C. and 37° C. is nearly identical at all shear rates).

[0424] Viscosity of rh collagen-methacrylamide: FIG. 4 is a comparative plot showing the viscosity of fibrillar rhcollagen-MA in DPBS at 4° C. (blue line) and 37° C. (red line), showing that the profiles are similar (both are shear thinning) but not identical, with zero shear rate values ​​of approximately 1000 cP at both temperatures.

[0425] Figure 5 is a comparative plot showing the thermal stability and, more importantly, the decrease in viscosity in the range of higher working temperatures (e.g., jetting temperatures) compared to the increase in viscosity with increasing temperature shown for bovine methacrylated settable collagen. Viscosity measurements were performed for 10 mg / ml rhcollagen-MA in 20 mM acetic acid (blue) and bovine settable collagen in acetic acid (red) at increasing temperature steps and recorded on a rheometer using the rotational temperature step CR mode at a speed of 5 l / s. The acetic acid solutions were prepared as described above.

[0426] [Example 3] Characterization of solidified rhcollagen-MA Photocrosslinking of rhcollagen-MA to measure loss and storage moduli: Rhcollagen-MA crosslinked scaffolds were formed in two different formulations for investigation in two separate experiments, respectively.

[0427] A 10% (v / v) stock solution of photoinitiator was prepared as follows: Irgacure 2959 was dissolved in a 1:1 solution of absolute ethanol / DDW to a final concentration of 100 mg / mL.

[0428] For the first preparation, 1-2 wt% fibrous rhcollagen-MA synthesized using a 10-fold molar excess of methacrylic reagent was dissolved in 0.1 M DPBS at room temperature, and then 0.1% Irgacure 2959 was added to a final volume of 1 mL of solution replica, which was then poured into a disk-shaped mold. A mercury light source was then used to illuminate the sample at 670 mW / cm. 2 The curing process was carried out for 7 and 10 seconds at an average intensity of 0.05g / ml from a distance of 1.5 cm to obtain crosslinked scaffolds.

[0429] The second formulation contained two different batches of fibrillar rhcollagen-MA synthesized with 15-fold and 20-fold molar excess of methacrylic reagent. 1-2 wt% was dissolved in 0.1 M DPBS and 0.1% Irgacure 2959 was added to a final volume of 1.5 mL. To obtain highly crosslinked scaffolds, a mercury light source was used, with 420 mW / cm 2 The curing process was carried out for 60 seconds from a distance of 2 cm with an average intensity of 0.01 mm.

[0430] Measurement of storage and loss modulus of scaffolds: The rheological behavior of rh collagen cross-linked disks was investigated by a parallel plate system using a PP20 sawtooth spindle and 20 mm sawtooth plate setup. The non-cross-linked rh collagen-MA was characterized using a C60 / 1°Ti cone-plate element.

[0431] To evaluate the rheological behavior of rhcollagen-MA, two sets of experiments were carried out separately.

[0432] The first set of oscillatory analyses over time at 37 °C are shown in Figure 6A and B. A 1 mL sample was subjected to an oscillatory force in controlled stress mode with a frequency of 1 Hz and a shear stress of 5 Pa for 300 seconds at 37 °C. Figure 6A shows the loss modulus / storage modulus (G' / G") and tan(delta) values ​​before UV curing, while Figure 6B shows these values ​​after light curing exposure (curing by crosslinking with the addition of a photoinitiator). The gap was adjusted to 90% of the original sample height to test the cured samples (1 ml disks). G' and G" values ​​were averaged over the range of 150-300 seconds for all measurements.

[0433] The results show that irradiation in the presence of a photoinitiator (irradiation-induced curing) increases the storage modulus of rhcollagen-MA by 2-fold. Furthermore, the results show that the scaffold properties can be controlled by varying the concentration of rhcollagen-MA and the irradiation time (irradiation; exposure to curing conditions). The large difference between the G' and G" values ​​of the crosslinked discs, and the tan(delta) values ​​close to zero, indicate elastic-like behavior.

[0434] In the second set, 1.5 mL cured (crosslinked) disks were characterized by vibration under a frequency sweep at 37 °C. G' was recorded at frequencies ranging from 0.01 to 100 Hz under a shear stress of 1 Pa. To start the measurements, 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.

[0435] Before any measurements were taken, the samples were kept on the plate covered with a humidity lid for 1 min to reach temperature equilibrium.

[0436] The results are shown in Figure 7 and show that the 1.5 mL disks irradiated for 60 seconds have higher G' values. The G' values ​​increase with increasing rhcollagen-MA concentration and increasing degree of methacrylation, indicating that the scaffold properties can be controlled.

[0437] In situ crosslinking of rhcollagen-MA: An appropriate amount of vehicle was added to the lyophilized material to prepare a 15 mg / ml solution of monomeric rhcollagen-MA in 10 mM HCl, and after complete dissolution, 0.1% Irgacure photoinitiator was added to each replicate.

[0438] In situ crosslinking of rhcollagen-MA was studied by applying an oscillatory force over time at 25°C using a strain of 7% and an angular frequency of 5 rad / s. 80 μl of the solution was placed in a 20 mm parallel plate geometry and the G' and complex viscosity properties were first measured before crosslinking as described above. An external UV 365 nm source was then introduced and the sample properties were recorded over time during the crosslinking reaction on the plate.

[0439] The results are shown in Figure 8. As shown in Figure 8, an immediate and rapid increase in the tested properties was observed over a short period of time, reaching a plateau in less than 30 seconds, indicating good curability of the formulation. After curing for 60 seconds, the light source was turned off and values ​​continued to be measured over time after cure, showing similar values, indicating stability.

[0440] [Example 4] Characterization of rhcollagen-MA formulations using different media and / or different additives Preparation of DMEM formulations: rhCollagen-Methacrylamide in DMEM: Lyophilized fibrillar rhcollagen-methacrylamide (prepared as described in Example 1 above, using a molar ratio of MA:rhcollagen of 15:1) was dissolved in DMEM medium to final concentrations of 20 mg / mL and 26 mg / mL.

[0441] 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.

[0442] Preparation of hyaluronic acid methacrylate (HAMA): 500 mg of HA was functionalized as described by Leach et al. [Leach et.al. 2002, Biotechnology and Bioengineering, vol. 82, no. 5]. That is, 1.8 mL of triethylamine, 1.8 mL of glycidyl methacrylate, and 1.8 g of tetrabutylammonium bromide were added separately to a 50 mL solution of 10 mg / mL HA in DDW, with the reaction mixture thoroughly mixed before each component was added. The resulting reaction mixture was stirred overnight at room temperature, after which HAMA was precipitated by adding 20 volumes of acetone, and then redissolved in DDW. This precipitation process was repeated twice to remove all possible by-products, and the resulting reaction product was freeze-dried.

[0443] rhCollagen-Methacrylamide / Hyaluronic Acid Methacrylate (HAMA) in DMEM: Hyaluronic acid methacrylate prepared as described above was added to the solution of fibrillar rhcollagen-MA to give a final concentration of 10 mg / mL HA-MA and 20 mg / mL rhcollagen-MA in DMEM medium.

[0444] Viscosity of DMEM formulation: The viscosity of rhcollagen-MA alone and in the presence of hyaluronic acid (HA) or HA-MA (prepared as described in Example 1) in DMEM at 25° C. was tested and the resulting data is shown in FIG.

[0445] As shown in Figure 9, the typical shear thinning behavior of rh collagen shown in Figures 2 and 3 is maintained for rh collagen-MA with or without the addition of HA / HA-MA. Increasing the concentration of rh collagen from 20 mg / mL to 26 mg / mL (green and red lines, respectively) increased the zero shear viscosity. The same is seen with the addition of 10 mg / mL of HA or HAMA (resulting in a final polymer concentration of 30 mg / mL).

[0446] Preparation of the formulation using 0.1M phosphate buffer 0.1M (pH 7.4) / NaCl 11.3 mM solution: The following stock solutions were prepared: 7.5 mg / ml monomeric rh collagen-methacrylamide (10x) in phosphate buffer (0.1 M) / saline (11.3 mM), 30 mg / ml PVAMA in phosphate buffer (0.1 M) / saline (11.3 mM), 30 mg / ml TiO2 in phosphate buffer (0.1 M) / saline (11.3 mM), and 30 mg / ml oxidized cellulose in phosphate buffer (0.1 M) / saline (11.3 mM). HAMA was purchased from Advanced BioMatrix and dissolved in 10 mM HCl to 10 mg / ml. 4.5 ml of HAMA solution was finally neutralized by adding 0.5 ml of 1 M phosphate buffer + 113 mM NaCl.

[0447] Preparation of monomeric rhcollagen-methacrylamide in phosphate buffer (0.1 M) / saline (11.3 mM): A stock solution of monomeric rhcollagen-methacrylamide was diluted to 5 mg / ml in 0.1 M phosphate buffer (pH 7.4) + 11.3 mM NaCl.

[0448] Preparation of rhcollagen-methacrylamide and HAMA in phosphate buffer (0.1 M) / saline (11.3 mM): Collagen MA (monomer rh collagen-methacrylamide ) and HAMA solutions (5:1 and 2:1 collagen MA:HAMA weight ratios) were prepared by mixing appropriate amounts of the above-mentioned collagen MA and HAMA stock solutions with phosphate buffer (0.1 M) / saline (11.3 mM) to give final concentrations of 5 mg / ml CollMA and 1 mg / ml HAMA (5:1 ratio) and 5 mg / ml CollMA and 2.5 mg / ml HAMA (2:1 ratio), respectively.

[0449] Preparation of rh collagen-methacrylamide and PVAMA in phosphate buffer (0.1 M) / saline (11.3 mM): Collagen MA (monomer) rh collagen-methacrylamide) and PVAMA (with collagen MA:PVAMA weight ratios of 5:1, 2:1, and 1:2) were prepared by mixing appropriate amounts of the above-mentioned collagen MA and PVAMA stock solutions with phosphate buffer (0.1 M) / saline (11.3 mM) to give final concentrations of 5 mg / ml CollMA and 1 mg / ml PVAMA (5:1 ratio), 5 mg / ml CollMA and 2.5 mg / ml PVAMA (2:1 ratio), and 5 mg / ml CollMA and 10 mg / ml PVAMA (1:2 ratio).

[0450] rh collagen-methacrylamide and titanium dioxide (TiO 2 Preparation of a solution of 11.3 mM phosphate buffer (0.1 M) / physiological saline (11.3 mM): Collagen MA (monomer rh collagen-methacrylamide Collagen MA and TiO2 (weight ratios of 5:1, 2:1, and 1:2) were prepared by mixing appropriate amounts of the above-mentioned collagen MA and TiO2 stock solutions with phosphate buffer (0.1 M) / saline (11.3 mM) to give final concentrations of 5 mg / ml CollMA and 1 mg / ml TiO2 (5:1 ratio), 5 mg / ml CollMA and 2.5 mg / ml TiO2 (2:1 ratio), and 5 mg / ml CollMA and 10 mg / ml TiO2 (1:2 ratio).

[0451] Preparation of a solution of rh collagen-methacrylamide and oxidized cellulose in phosphate buffer (0.1 M) / saline (11.3 mM): Collagen MA (monomer rh collagen-methacrylamide ) and oxidized cellulose (with collagen MA:oxidized cellulose weight ratios of 5:1, 2:1, and 1:2) were prepared by mixing appropriate amounts of the collagen MA and oxidized cellulose stock solutions described above with phosphate buffer (0.1 M) / saline (11.3 mM) to give final concentrations of 5 mg / ml CollMA and 1 mg / ml oxidized cellulose (5:1 ratio), 5 mg / ml CollMA and 2.5 mg / ml oxidized cellulose (2:1 ratio), and 5 mg / ml CollMA and 10 mg / ml oxidized cellulose (1:2 ratio).

[0452] Viscosity of formulations using 0.1M Phosphate Buffer 0.1M (pH 7.4) / NaCl 11.3mM: The viscosity of formulations (collagen-MA:additive ratios of 5:1, 2:1 and 1:2) containing 5 mg / ml rh-collagen methacrylamide (rh-collagen-MA; col) in 0.1 M phosphate buffer (pH 7.4) / 11.3 mM NaCl (physiological osmolality) and enriched with various additives (polyvinyl alcohol methacrylate (PVAMA), hyaluronic acid methacrylate (HAMA), titanium dioxide (TiO2) and oxidized cellulose (OC)), prepared as described above, was tested. Measurements were performed at T=22 °C using a cone-on-plate configuration (spindle C35 / 1 Ti Thermo HAAKE Rheo Stress600 instrument). The obtained data are shown in Figures 10 to 13.

[0453] FIG. 10 is a comparative plot showing the viscosity of 5 mg / ml rh collagen-MA (col, black curve) and 5 mg / ml rh collagen-MA + polyvinyl alcohol methacrylate (PVAMA, green curve) with rh collagen-MA:PVAMA weight ratios of 5:1 (green solid curve), 2:1 (green dashed curve), and 1:2 (green dotted curve).

[0454] FIG. 11 is a comparative plot showing the viscosity of 5 mg / ml rhcollagen-MA (col, black curve) and 5 mg / ml rhcollagen-MA+HAMA (blue curve) with rhcollagen-MA:HAMA weight ratios of 5:1 (blue solid curve) and 2:1 (blue dashed curve).

[0455] FIG. 12 is a comparative plot showing the viscosity of 5 mg / ml rhcollagen-MA (col, black curve) and 5 mg / ml rhcollagen-MA+TiO2 (red curve) with rhcollagen-MA:HAMA weight ratios of 5:1 (red solid curve), 2:1 (red dashed curve), and 1:2 (red dotted curve).

[0456] FIG. 13 is a comparative plot showing the viscosity of 5 mg / ml rh collagen-MA (col, black curve) and 5 mg / ml rh collagen-MA + oxidized cellulose (orange curve) with rh collagen-MA:HAMA weight ratios of 5:1 (orange solid curve), 2:1 (orange dashed curve), and 1:2 (orange dotted curve).

[0457] Cured rhcollagen-MA formulation with 0.1M phosphate buffer 0.1M (pH 7.4) / NaCl 11.3mM: The above formulations containing rhcollagen-MA alone or with various additives (2:1 collagen:additive weight ratio) were mixed with the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (0.1%). 130 μL of the formulations were introduced into a silicon mold (8 mm × 8 mm × 8 mm) and irradiated with UV light (365 nm) at a distance of 1.5 cm for 20 s.

[0458] An image of the resulting molded object is shown in FIG.

[0459] Viscosity of rhcollagen-MA formulations using phosphate buffer and HCl and G' measurements of hardened objects formed therefrom: A 10 mg / ml solution of monomeric rhcollagen-MA (10x) in phosphate buffer 0.1M / NaCl 15.8 mM (approximately 16 mM) or 10 mM HCl was prepared by adding the appropriate amount of medium to the lyophilized material. After complete dissolution, the viscosity of the formulation was tested and then allowed to harden.

[0460] The viscosities of these formulations at high frequency were recorded at room temperature by a rheometer using rotational CR mode, and the data obtained are shown in Figure 15. As shown in Figure 15, the viscosity of the 0.1 M phosphate buffer / 0.016 M NaCl (total 300 mOs / L) formulation (blue line) is higher than 10 mM HCl (red line) and 20 mM acetic acid (data not shown). The shear thinning behavior of this phosphate buffer medium is more pronounced compared to all other tested media.

[0461] These formulations were cured by a UV LED source at 365 nm. 1.6 ml of a solution containing 0.1% photoinitiator was poured into a disk mold and 100 mW / cm 2 The cured bodies were then cured for 90 seconds by RT and the high frequency G' values ​​of the cured bodies were measured at 37°C using a rheometer in oscillatory CR mode.

[0462] The resulting data is shown in Figure 16 and demonstrates the high storage modulus of the cured bodies formed with the phosphate buffer-based formulations.

[0463] [Example 5] A 20 mg / ml solution of monomeric rhcollagen-MA (prepared using a 10-fold molar excess of MA) in Hepes-buffered saline was mixed with N-vinylpyrrolidinone and Eosin Y / TEA (triethanolamine) as a photoinitiator. 120 microliters was dispensed into a silicon mold (8 mm x 8 mm x 3 mm) and covered with a mouse skin patch (Science in Action Ltd.). The formulation was transdermally polymerized by irradiating the exterior of the skin patch with white light from a white LED torch for 6 minutes.

[0464] FIG. 17 is a photograph of the skin patch after irradiation taken using a camera, which shows that rhcollagen-MA has polymerized and incorporated into the skin tissue.

[0465] [Example 6] Monomeric rhcollagen-MA alone and in combination with calcium phosphate particles were evaluated in a cell proliferation assay using normal human dermal fibroblasts (Promocell).

[0466] Objects (scaffolds) were prepared from freeze-dried rhcollagen-MA dissolved in DDW containing (1) rhcollagen-MA alone (9 mg / ml), (2) rhcollagen-MA (9 mg / ml) and nanohydroxyapatite (nanoHA) (7 mg / ml), and (3) rhcollagen-MA (9 mg / ml) and nanotricalcium phosphate (nanoTCP) (7 mg / ml). Each solution was UV cured, freeze-dried, and sterilized with ethylene oxide (ETO). Normal human dermal fibroblasts were seeded on the scaffolds and cell proliferation was measured by a colorimetric assay (WST-1) on days 1, 3, and 7. Figure 18 shows the data obtained, which indicates good cell proliferation in all tested formulations.

[0467] While the present invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0468] All publications, patents, and patent applications mentioned in this specification are incorporated herein in their entirety to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated herein. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not necessarily be construed as limiting. [Sequence List Free Text]

[0469] SEQ ID NO: 11: Synthetic sequence comprising the coding region of the vascular signal sequence of the barley gene thiol protease gene aleurain precursor fused to the human prolyl 4-hydroxylase β subunit and its adjacent regions. SEQ ID NO: 12: Synthetic sequence comprising the coding region of the vascular signal sequence of the barley gene thiol protease gene aleurain precursor fused to the human prolyl 4-hydroxylase β subunit and its adjacent regions. SEQ ID NO: 13: Synthetic sequence comprising the coding region of the vascular signal sequence of the barley gene thiol protease gene aleurain precursor fused to human lysyl hydroxylase 3 and its adjacent regions. SEQ ID NO: 14: Vacuolar targeting sequence of the thiol protease gene aleurain precursor (NCBI accession number P05167 GI:113603)

Claims

1. 1. A process for additive manufacturing of a three-dimensional object, characterized in part by a collagen-based material, comprising successively forming a plurality of layers in a constituent pattern corresponding to a shape of the object, comprising: at least a portion of the plurality of layers are formed from a modeling material formulation comprising a plant-derived recombinant human collagen having at least one hardenable group that polymerizes, entangles, and / or crosslinks upon exposure to radiation, and further comprising an aqueous carrier, the modeling material formulation having a pH within the range of 6±10% to 8±10%; This process produces three-dimensional objects.

2. The process of claim 1, further comprising exposing a portion of the plurality of layers to radiation.

3. The process of claim 1 or 2, wherein the at least one curable group comprises a (meth)acrylic group.

4. The process according to any one of claims 1 to 3, wherein the plant-derived recombinant human collagen is plant-derived recombinant human type I collagen.

5. 5. The process of any one of claims 1 to 4, wherein the concentration of the plant-derived recombinant human collagen with curable groups in the modeling material formulation ranges from 0.5 mg / mL to 50 mg / mL or from 0.5 mg / mL to 20 mg / mL.

6. 6. The process of any one of claims 1 to 5, wherein the modeling material formulation has a viscosity of 2,000 centipoise or less or 1,500 centipoise or less at zero shear rate and 37°C or at room temperature with a shear rate of 5 1 / sec, and the concentration of the recombinant human collagen with curable groups is at least 3 mg / mL.

7. 7. The process of any one of claims 1 to 6, wherein at least a portion of said plurality of layers are formed by a modeling material formulation comprising at least one hardenable material other than said plant-derived recombinant human collagen having said hardenable groups.

8. 8. The process of claim 7, wherein the modeling material formulation comprises the plant-derived recombinant human collagen having the curable groups and the at least one curable material.

9. 9. The process of claim 8, wherein the weight ratio of said plant-derived recombinant human collagen having said hardenable groups to said at least one hardenable material is in the range of 10:1 to 1:

2.

10. 9. The process of claim 8, wherein the weight ratio of said plant-derived recombinant human collagen having said hardenable groups to said at least one hardenable material is in the range of 10:1 to 1:

1.

11. 9. The process of claim 8, wherein the weight ratio of said plant-derived recombinant human collagen having said hardenable groups to said at least one hardenable material is in the range of 5:1 to 2:

1.

12. The process according to any one of claims 1 to 11, wherein at least a portion of the plurality of layers is formed by a modelling material formulation comprising a reagent for modifying the mechanical and / or rheological and / or physical properties of the formulation and / or parts of an object constructed from the formulation.

13. The process of any one of claims 1 to 12, wherein at least a portion of said plurality of layers is formed by a modeling material formulation comprising a biological material other than said plant-derived recombinant human collagen.

14. The process of claim 13, wherein the modeling material formulation comprises the plant-derived recombinant human collagen and biological material.

15. The process of any one of claims 1 to 14, wherein the modeling material formulation upon solidification has a storage modulus (G') of at least 1,000 Pa.

16. The process of any one of claims 1 to 15, wherein the modeling material increases its storage modulus (G') by at least 10 times upon solidification.

17. The process of claim 1, wherein the temperature at which the layer is formed is at least 20°C.

18. The process of claim 1, wherein the temperature at which the layer is formed is between 10°C ± 10% and 37°C ± 10%.

19. The process of claim 1, wherein the temperature at which the layer is formed is between 20°C ± 10% and 37°C ± 10%.

20. The process of claim 1, wherein the temperature at which the layer is formed is between 20°C ± 10% and 30°C ± 10%.

21. The process of claim 1, wherein the temperature at which the layer is formed is between 20°C ± 10% and 25°C ± 10%.

22. The process according to any one of claims 1 to 21, wherein the three-dimensional biological object is for the repair of damaged tissue.

23. The process according to any one of claims 1 to 21, wherein the three-dimensional biological object is for use as an artificial tissue or organ.

24. 1. A formulation for additive manufacturing of three-dimensional biological objects comprising at least a portion of a collagen-based material, the formulation comprising a plant-derived recombinant human collagen having at least one hardenable group that polymerizes, entangles, and / or crosslinks upon exposure to radiation and an aqueous carrier, wherein when the concentration of the plant-derived recombinant human collagen having hardenable groups is at least 3 mg / mL, the formulation has a pH in the range of 6±10% to 8±10%, and the formulation has a viscosity of 2,000 centipoise or less or 1,500 centipoise or less under conditions of a shear rate of 5 1 / sec at room temperature and / or at zero shear rate at 37°C.

25. 25. The formulation of claim 24, wherein the three-dimensional biological object is for repair of damaged tissue.

26. 25. The formulation of claim 24, wherein the three-dimensional biological object is for use as an artificial tissue or organ.

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