Collagen-containing hardening formulation

Abstract

Conjugates formed of collagen and multiple curable elastomeric moieties covalently attached thereto, curable formulations (e.g., bio-ink compositions) including the conjugates, and additive manufacturing of three-dimensional objects using the curable formulations are provided. Also provided is a method / process for additive manufacturing using collagen having multiple photocurable groups, in which the amount of photoinitiator mixed with the collagen determines the viscosity of the collagen-containing formulation.

Description

[Technical field]

[0001] Related Applications This application claims priority under §119(e) of U.S. Provisional Patent Application No. 63 / 272,313, filed October 27, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing A file entitled 94209.xml, created on October 17, 2022, consisting of 5,926,912 bytes, submitted concurrently with this application is hereby incorporated by reference as if it were a part of this specification.

[0003] FIELD AND BACKGROUND OF THEINVENTION 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]

[0004] 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].

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] For example, in a three-dimensional printing process, a build material is dispensed from a dispensing head having a set or array of nozzles to deposit a layer onto a receiving substrate, and depending on the build material, the layer can then be cured or solidified using an appropriate device.

[0010] The build materials may include a modeling material formulation and a support material formulation, which upon solidification respectively form the object and a temporary support structure that supports the object being built. The modeling material formulation is deposited to produce the desired object. The support material formulation, with or without modeling material elements, is also used to provide support structures in certain areas of the object being built to ensure proper vertical alignment of subsequent object layers. This is done, for example, when the object includes overhangs or shapes (e.g., curved shapes, negative angles, gaps, etc.).

[0011] Both the modeling and support material formulations typically exhibit a viscosity that allows dispensing / deposition and, in some cases, a higher viscosity upon dispensing and, in some cases, exposure to curing / solidifying conditions. Both the modeling and support materials are preferably liquid at the dispensing operating temperature and then solidify to form the required layer shape upon exposure to solidifying or curing conditions, typically curing energy (e.g., UV curing). 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, digital light processing, 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 / gelation 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 has been 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 [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 (meth)acrylates have been extensively characterized for their 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, many factors hinder the use of collagen (meth)acrylate 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 of the present application has developed a 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 WO 2006 / 035442, WO 2009 / 053985, WO 2011 / 064773, WO 2013 / 093921, WO 2014 / 147622, ​​and patents and patent applications derived therefrom, the entire contents of all of which are hereby incorporated by reference.

[0024] The assignee of the present application, WO 2018 / 225076, describes formulations and kits containing the same that can be used in the preparation of or as modeling material formulations for additive manufacturing of 3D objects (e.g., 3D bioprinting). The formulations exhibit a desired viscosity at temperatures above 10° C. (e.g., room temperature or 37° C.), allowing additive manufacturing to be performed without cooling the system or parts thereof.

[0025] Further background art includes U.S. Patent Application Publication No. 2018 / 0193524, WO 2015 / 032985, Drzewiecki et al. (2014) Langmuir, 30(37), 11204-11211, Ravichandran et al. (2015) Journal of Materials Chemistry B, 4(2), 318-326, and Gaudet & Shreiber (2012) Biointerphases, 7(1), 25.

[0026] Further background art includes Zhang et al., Burns Trauma. 2022; 10: tkac010, International Publication No. 2022 / 093236, U.S. Patent Application Publication Nos. 2020 / 339925 and 2021 / 229364, U.S. Patent No. 10,597,289, and CN114958079. Summary of the Invention

[0027] According to one aspect of some embodiments of the present invention, there is provided a conjugate comprising collagen and a plurality of elastic / elastomeric moieties covalently bonded to the collagen, at least a portion of the elastic / elastomeric moieties having curable groups.

[0028] According to some of the embodiments described herein, a curable group is at the end of each of the elastic / elastomeric portions.

[0029] According to some of the embodiments described herein, the hardenable group is a photohardenable group or a photopolymerizable group.

[0030] According to some of the embodiments described herein, the curable group is a (meth)acrylic group.

[0031] According to some of the embodiments described herein, at least a portion of the elastic / elastomeric portion is a poly(alkylene glycol)-containing portion.

[0032] According to some of the embodiments described herein, at least a portion of the or each of the elastic / elastomeric segments comprises a poly(alkylene glycol) segment terminated with an acrylic or (meth)acrylic group.

[0033] According to some of the embodiments described herein, at least a portion of the elastic / elastomeric portion is covalently bonded to lysine residues of collagen.

[0034] According to some of the embodiments described herein, at least 1%, such as 1-20% or 1-10% of the lysine residues in the collagen have a resilient / elastomeric moiety covalently attached thereto.

[0035] According to some of the embodiments described herein, at least a portion of the elastic / elastomeric moieties are attached to lysine residues via carbamate bonds.

[0036] According to some of the embodiments described herein, the collagen has a plurality of curable groups, for example photocurable groups (in addition to the curable elastic / elastomeric moieties).

[0037] According to some of the embodiments described herein, the collagen is human type I collagen.

[0038] According to some of the embodiments described herein, the collagen is recombinant collagen.

[0039] According to some of the embodiments described herein, the collagen is recombinant collagen of plant origin.

[0040] According to some of the embodiments described herein, the collagen is plant-derived recombinant human type I collagen.

[0041] According to one aspect of some embodiments of the present invention there is provided a curable formulation comprising a conjugate as described herein in any of the embodiments and any combination thereof.

[0042] According to some of the embodiments described herein, the curable formulation further comprises an aqueous carrier.

[0043] According to some of the embodiments described herein, the concentration of the conjugate ranges from 0.5 mg / mL to 50 mg / mL, or from 0.5 mg / mL to 20 mg / mL, or from 0.5 mg / mL to 10 mg / mL, or from 1 mg / mL to 10 mg / mL.

[0044] According to some of the embodiments described herein, the curable formulation further comprises at least one additional curable material.

[0045] According to some of the embodiments described herein, the additional material has a curable (eg, photocurable) group.

[0046] According to some of the embodiments described herein, the additional material is or includes a poly(alkylene glycol) terminated with at least one (meth)acryl group.

[0047] According to some of the embodiments described herein, the concentration of the additional hardenable material ranges from 1 to 20 wt. %, or 1 to 10 wt. %, of the total weight of the formulation.

[0048] According to some of the embodiments described herein, the hardenable composition further comprises a biological material other than the hardenable collagen.

[0049] According to some of the embodiments described herein, the curable formulation further comprises an agent that promotes polymerization of the conjugate.

[0050] According to some of the embodiments described herein, the curable group is a photocurable group and the reagent is a photoinitiator.

[0051] According to some of the embodiments described herein, the curable formulation further comprises a pigment material capable of absorbing light at wavelengths between 300 nm and 800 nm or between 300 and 450 nm.

[0052] According to some of the embodiments described herein, the dye material has a plurality of negatively charged groups.

[0053] According to some of the embodiments described herein, the pigment substance is vitamin B12.

[0054] According to some of the embodiments described herein, the dye substance is minocycline.

[0055] According to some of the embodiments described herein, the dye substance is a quinoline.

[0056] According to some of the embodiments described herein, the amount of color material ranges from 0.01 to 5% by weight of the total weight of the composition as described in any of the embodiments and any combination thereof herein.

[0057] According to one aspect of some embodiments of the present invention, there is provided a process for additive manufacturing of a three-dimensional object characterized at least in part 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 a hardenable formulation as described herein in any of the embodiments and any combination thereof, thereby producing the three-dimensional object.

[0058] According to some of the embodiments described herein, the process further includes exposing at least a portion of the plurality of layers to suitable curing conditions to solidify the curable formulation.

[0059] According to some of the embodiments described herein, for at least a portion of the multiple layers, 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 comprised of the formulation.

[0060] According to some of the embodiments described herein, the dispensing for at least a portion of the plurality of layers is a further dispensing of a modeling material formulation that includes a biological material other than human recombinant collagen.

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

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

[0063] According to some of the embodiments described herein, the three-dimensional biological object is used to repair damaged tissue.

[0064] According to some of the embodiments described herein, the three-dimensional biological objects are used as artificial tissues or organs.

[0065] According to an aspect of some embodiments of the present invention, there is provided a curable formulation for additive manufacturing of three-dimensional objects, the formulation comprising a photocurable biological material and a pigment substance capable of absorbing light at a wavelength between 300 nm and 800 nm, the pigment substance comprising vitamin B12.

[0066] According to some of the embodiments described herein, the amount of pigment material ranges from 0.01 to 5% by weight of the total weight of the composition.

[0067] According to some of the embodiments described herein, the photocurable biological material includes collagen having a plurality of photocurable groups.

[0068] According to some of the embodiments described herein, the photocurable group comprises a (meth)acryl group.

[0069] According to some of the embodiments described herein, the photocurable groups are directly attached to the collagen.

[0070] According to some of the embodiments described herein, the collagen comprises a plurality of elastic / elastomeric moieties covalently attached thereto, at least a portion of the elastic moieties having photocurable groups.

[0071] According to some of the embodiments described herein, the collagen is human type I collagen, such as recombinant human collagen, such as recombinant human type I collagen of plant origin.

[0072] According to one aspect of some embodiments of the present invention, there is provided a process for additive manufacturing of a three-dimensional object, characterized in that at least a portion of the object is composed of a biological 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 the dispensing for at least a portion of the plurality of layers is a modeling material formulation comprising a vitamin B12-containing curable formulation as described in any of the embodiments and any combination thereof herein, thereby producing a three-dimensional object.

[0073] According to some of the embodiments described herein, the process further includes exposing a portion of the plurality of layers to radiation suitable to solidify the formulation.

[0074] According to one aspect of some embodiments of the present invention, there is provided a process for additive manufacturing of a three-dimensional object characterized in that the object is at least in part comprised of a collagen-based material, the process comprising: Selecting additive manufacturing technologies; preparing a modeling material formulation (e.g., a bio-ink composition) by combining collagen having at least a plurality of photocurable groups, a photoinitiator, optionally an aqueous carrier, and optionally other curable and / or non-curable components, wherein the amount of photoinitiator is selected to provide a suitable viscosity for dispensing the formulation in an additive manufacturing technique; and dispensing at least one modeling material formulation to successively form a plurality of layers in a configuration pattern corresponding to the shape of the object; Including, dispensing at least a portion of the plurality of layers is dispensing a modeling material formulation including collagen having a plurality of photocurable groups; This provides a process for manufacturing three-dimensional objects.

[0075] According to some of the embodiments described herein, the collagen is human type I collagen as described herein in any of the embodiments and any combination thereof.

[0076] According to some of the embodiments described herein, the photocurable group comprises a (meth)acryl group.

[0077] According to some of the embodiments described herein, the photoinitiator is an acylphosphine oxide type photoinitiator.

[0078] According to some of the embodiments described herein, the photoinitiator is phenyl-2,4,6-trimethylbenzoylphosphine oxide or a salt thereof.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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:

[0083] The drawings are as follows: [Brief description of the drawings]

[0084] [Figure 1] FIG. 1 is a simplified schematic depiction of (meth)acrylated PEG moieties conjugated to lysine residues of collagen (recombinant human collagen type I) according to some embodiments of the present invention. [Diagram 2] 1 shows an SDS-PAGE comparing collagen, collagen-methacrylated PEG conjugate (CPM), and a reference mixture of proteins of known molecular weights as indicated ("ladder"). [Diagram 3] FIG. 3 shows plots showing the viscosity of a formulation containing collagen-methacrylated PEG conjugate (CPM, FIG. 3A) and a formulation containing methacrylated collagen (CMR, FIG. 3B) as a function of shear rate after relaxation for 1, 3, or 5 minutes. [Figure 4] The recovery of the viscosity of a formulation containing collagen-methacrylated PEG conjugate (CPM, FIG. 4A) and a formulation containing methacrylated collagen (CMR, FIG. 4B) upon manipulation of shear force is shown, demonstrating the recovery of the tested formulations. [Diagram 5] FIG. 1 shows a comparative plot illustrating the storage modulus (G') of a formulation containing methacrylated rh collagen (CMR), a formulation containing an exemplary methacrylated collagen-PEG conjugate according to some of the present embodiments (CPM), and a formulation lacking protein. [Figure 6]Photographs of a solution of methacrylated collagen (vial on the left) and an exemplary solution of a methacrylated collagen-PEG conjugate according to some of the present embodiments (vial on the right) upon addition of a polysulfate dye are shown. [Figure 7] 1 shows a comparative plot showing the viscosity at various shear rates of CMR formulations containing Vitamin B12 or 4-nitrophenol as a dye. [Figure 8] 1 shows comparative plots illustrating the effect of the amount of photoinitiator (LAP) on the viscosity of formulations containing CMR. [Figure 9] 1 shows a comparative plot illustrating the storage modulus (G') of formulations containing methacrylated rh-collagen (CMR, 5 mg / mL), LAP (0.5 wt %), and various concentrations of minocycline. [Figure 10] FIG. 1 shows a schematic diagram of the synthesis of collagen (recombinant human type I collagen) bearing multiple (meth)acrylic acid groups and multiple PEG moieties conjugated to lysine residues (lower scheme) and intermediate reagents used in its preparation (upper scheme). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] 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.

[0086] 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 or of being practiced or carried out in various ways.

[0087] 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, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0088] In investigating curable collagen materials that exhibit improved mechanical properties and can be beneficially utilized in bio-ink compositions that can be used for additive manufacturing of collagen-containing three-dimensional objects, the inventors have conceived, successfully prepared, and demonstrated conjugates that include collagen having curable elastic / elastomeric moieties attached thereto. An example of such a conjugate is illustrated diagrammatically in FIG. 1. The inventors have shown that formulations that include such conjugates exhibit improved recovery under shear and improved modulus, as shown in FIGS. 3A-B and 4A-B, exhibit improved properties of the solidified material, as shown in FIG. 5, and further enable the use of absorbing dye additives bearing negatively charged groups without adversely affecting the consistency of the formulation, as illustrated in FIG. 6.

[0089] Embodiments of the present invention relate to conjugates of collagen and curable elastic moieties, bio-ink compositions comprising such conjugates, and their use in additive manufacturing.

[0090] In the course of testing bio-ink compositions including curable collagen, the inventors further discovered that vitamin B12 can be advantageously used as an absorbing dye substance in such bio-ink compositions, as shown in FIG.

[0091] In the course of testing bio-ink compositions containing curable collagen, the inventors further discovered that minocycline can be advantageously used as an absorbing dye substance in such bio-ink compositions, as shown in FIG.

[0092] Accordingly, embodiments of the present invention further relate to curable formulations (e.g., collagen-based bio-ink compositions described herein) that include vitamin B12 as an absorbing dye substance as described herein.

[0093] The inventors have further discovered that the curable collagens described herein interact with a common class of photoinitiators (acylphosphine oxide type photoinitiators such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)) such that the amount of photoinitiator affects the viscosity (prior to exposure to curing radiation) of a curable formulation containing it, as illustrated in FIG. 8.

[0094] Accordingly, embodiments of the present invention further relate to curable formulations that include variable amounts of photoinitiators and that can be appropriately designed and / or selected to suit the respective additive manufacturing methods described herein.

[0095] collagen: The conjugate according to this embodiment comprises collagen to which a hardenable elastic moiety is attached.

[0096] 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.

[0097] Collagen according to some of the embodiments 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 a collagen sequence as set forth in Table A as determined by the BlastP software of the National Center for Biotechnology Information (NCBI) using default parameters). Homologs can also refer to deletion, insertion or substitution variants thereof (including amino acid substitutions) and biologically active polypeptide fragments thereof.

[0098] According to a particular embodiment, the collagen is human collagen.

[0099] In another embodiment, the collagen comprises the naturally occurring amino acid sequence of human collagen.

[0100] Table A below provides examples of NCBI sequence numbers for collagen. Table A NCBI SEQ ID NOs for Exemplary Procollagens P02452 1 P08123 2

[0101] 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

[0102] 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

[0103] According to one embodiment, the collagen contains sufficient telopeptides to enable it to form fibrils under appropriate conditions.

[0104] Thus, for example, the collagen can be atelocollagen, telocollagen or procollagen.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] According to other embodiments, the collagen lacks its telopeptides and is unable to undergo fibrillogenesis.

[0109] According to another embodiment, the collagen is a mixture of the collagen types mentioned above.

[0110] According to certain embodiments, the collagen is genetically engineered by recombinant DNA techniques (eg, human collagen).

[0111] 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) that disrupt intermolecular bonds and remove immunogenic non-helical telopeptides without affecting the basic rigid triple helical structure that confers collagen's desirable properties. (See U.S. Patent Nos. 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037; 3,949,073; 4,233,360; and 4,488,911 for general methods 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.

[0112] Plants expressing collagen chains and procollagens are known in the art (see, e.g., WO06 / 035442; 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.

[0113] It will be understood that embodiments of the present invention also contemplate genetically modified collagens / atelocollagens, such as collagenase-resistant collagens, etc. (see, e.g., Wu et al., Proc Natl. Acad Sci, Vol. 87, p.5888-5892, 1990).

[0114] 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.

[0115] 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.

[0116] Recombinant procollagen is usually produced by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen.

[0117] Exemplary polynucleotide sequences encoding human procollagen are shown in SEQ ID NOs:7, 8, 9 and 10.

[0118] Human telocollagen is usually produced by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen and at least one exogenous polynucleotide sequence encoding the relevant protease. Alternatively, the protease can be added after isolation of the recombinant procollagen.

[0119] 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 collagens containing hydroxyproline only at the Y position of the Gly-XY triplet requires the co-expression of collagens and human or mammalian P4H genes [Olsen et al, Adv Drug Deliv Rev. 2003 Nov 28;55(12):1547-67].

[0120] Thus, according to one embodiment, procollagen or telocollagen is expressed in an intracellular compartment of a plant that does not have endogenous P4H activity.

[0121] 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, an apoplast, or a chloroplast. According to a particular embodiment, the subcellular compartment is a vacuole.

[0122] According to other embodiments, the intracellular compartment is the apoplast.

[0123] Accumulation of expressed procollagen in intracellular compartments that lack endogenous P4H activity can be achieved by any one of several approaches.

[0124] 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).

[0125] 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.

[0126] Alternatively, the sequence of the procollagen can be modified in a way that alters the cellular localization of the procollagen when expressed in plants.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] Thus, genetically modified cells according to some embodiments 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.

[0132] An LH3 coding sequence such as that shown in SEQ ID NO:13 may be used for such purposes.

[0133] 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.

[0134] 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.

[0135] Additionally, there are several methods by which nucleic acid constructs can be directly introduced into the DNA of DNA-containing organelles such as chloroplasts.

[0136] 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. (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, including direct uptake of DNA into protoplasts: Toriyama, K. et al. (1988) Bio / Technology 6:1072-1074. 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 tissues by particle bombardment: Klein et al. Bio / Technology (1988) 6:559-563; McCabe et al. Bio / Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209. by 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.

[0137] 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.

[0138] 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).

[0139] 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.

[0140] 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.

[0141] 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.

[0142] The proteases used to cleave recombinant propeptide- or telopeptide-containing collagen are not necessarily 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).

[0143] In some of the embodiments described herein, the recombinant human collagen is recombinant human type I collagen.

[0144] In some embodiments described herein, the recombinant human collagen is a plant-derived recombinant human collagen, and in some embodiments, the plant is tobacco. Exemplary collagens are described in Stein H. (2009) Biomacromolecules;10:2640-5, WO 2006 / 035442, WO 2009 / 053985, WO 2011 / 064773, WO 2013 / 093921, and WO 2014 / 147622.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] In some of the embodiments described herein, the human recombinant collagen (rhcollagen) described in any of the embodiments herein is monomeric rhcollagen.

[0149] By "monomeric" is meant rh collagen as described herein that is soluble in aqueous solution and does not form fibrillar aggregates.

[0150] In some of the embodiments described herein, the human recombinant collagen (rh collagen) described in any of the embodiments herein is fibrillar rh collagen.

[0151] By "fibrous" is meant rh collagen as described herein that assumes 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 WO 2018 / 225076.

[0152] Sclerosing collagen: Some embodiments of the present invention relate to sclerotic collagen.

[0153] "Curable" as used herein means a material that is capable of hardening or solidifying (eg, changing viscosity or G') as defined herein upon exposure to appropriate curing conditions.

[0154] Curable materials typically harden or harden through polymerization and / or crosslinking.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] In some of the embodiments described herein, the curable material can be a monofunctional curable material or a multifunctional curable material.

[0159] 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).

[0160] 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.

[0161] "Sclerotic collagen" refers to collagen as described herein in any of 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.

[0162] The terms "sclerotic collagen," "sclerotic rh collagen," "collagen having one or more (or at least one) sclerotic groups," and "rh collagen having one or more (or at least one) sclerotic groups" are used interchangeably herein.

[0163] 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.

[0164] According to some of the embodiments described herein, the sclerotic collagen is as described in WO 2018 / 225076.

[0165] According to some of the embodiments described herein, the hardenable collagen refers to a collagen as described herein (e.g., rh collagen as described herein in any of the embodiments) to which one or more hardenable groups are either directly attached (e.g., by covalent bonds to each lysine residue of the collagen) or are not attached by an elastic moiety that terminates in a hardenable group as described herein. However, it should be noted that the hardenable collagen can further include one or more elastic moieties that terminate in a hardenable group, as described herein in connection with conjugates.

[0166] 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.

[0167] In some embodiments, the curable groups are capable of polymerizing and / or crosslinking via a free radical mechanism.

[0168] Examples of such curable groups include acrylic groups, such as acrylic acid groups, methacrylic acid groups, acrylamide groups, and methacrylamide groups, collectively referred to herein as (meth)acrylic groups. Other free radically curable groups can include thiols, vinyl ethers, and other groups characterized by a reactive double bond.

[0169] 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.

[0170] Other hardenable groups can include, for example, the formation of an amide bond between a functional carboxylic acid and an amine group (each being a hardenable group that can react with the other to crosslink), the formation of an imine bond between an amine and an aldehyde group, 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.

[0171] Any other hardenable groups are contemplated.

[0172] 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.

[0173] 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.

[0174] When the hardenable collagen contains more than one hardenable group, the hardenable groups may be the same or different.

[0175] 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.

[0176] According to some of the embodiments described herein, the hardenable group is a photohardenable group or a photopolymerizable group (eg, a (meth)acrylic group such as an acrylate or methacrylate).

[0177] Alternatively or additionally, the curable groups are thiol-containing groups, which upon curing result in disulfide cross-links.

[0178] Alternatively or additionally, the hardenable groups or moieties are hardened by chemical reaction such as glycosylation or conjugation (using a coupling agent such as EDC).

[0179] According to some embodiments, the hardenable groups include amine and carboxyl groups that form peptide bonds upon curing.

[0180] 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 (meth)acrylic groups as defined herein.

[0181] 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).

[0182] 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).

[0183] 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.

[0184] 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.

[0185] In some embodiments, at least 20%, or at least 30%, or at least 40%, or 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 10%-90%, or 10%-80%, or 10%-60%, or 10-50%, or 20-90%, or 20-80%, or 20-60%, or 20-50% of its lysine residues (including intermediate values ​​and subranges therebetween) are substituted with methacrylamide or acrylamide groups.

[0186] The hardenable collagen (e.g., rh collagen) described herein can be prepared by reacting a material that contains or generates hardenable groups with collagen (e.g., rh collagen), for example as described in WO 2018 / 225076.

[0187] The number of hardenable groups in the collagen (eg, rh collagen) can be controlled by manipulating the amount of material that reacts with the collagen (eg, rh collagen) to generate hardenable groups.

[0188] 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.

[0189] Conjugates: According to an aspect of some embodiments of the present invention there is provided a conjugate comprising collagen and a plurality of hardenable elastic moieties covalently bonded to the collagen.

[0190] Conjugates according to embodiments of this aspect of the invention can be considered as hardenable collagens, which upon exposure to curing conditions solidify, e.g., by polymerization and / or crosslinking as described herein, of at least the hardenable elastic moieties, and possibly other hardenable groups on the collagen (if the hardenable collagen is conjugated to multiple hardenable elastic moieties).

[0191] According to some of the embodiments described herein, the conjugate comprises collagen and a plurality of elastic moieties covalently bonded to the collagen, at least some of the elastic moieties having hardenable groups as defined herein.

[0192] By "multiple" it is meant two or more moieties, preferably three or more, attached to the collagen.

[0193] The terms "elastic" and "elastomeric" are used interchangeably herein when referring to a group (eg, a curable group) or a material (eg, a curable material).

[0194] The elastomeric segments may be the same or different, and if different, the difference may be in the chemical composition or stereochemistry of the elastomeric segments, and / or the type of curable group, and / or the location of the curable group.

[0195] The elastic segment having a curable group is also referred to herein interchangeably as a "curable elastic segment" or a "curable elastomeric segment" or an "elastomeric segment having a curable group" or an "elastomeric segment having a curable group," all meaning that the elastic or elastomeric segment has one or more curable groups.

[0196] According to some of the embodiments described herein, elastomeric moieties are those moieties that impart elasticity to the solidified material formed upon polymerization and / or crosslinking of each curable material. Such moieties typically include alkyl, alkylene chains, hydrocarbon chains, alkylene glycol groups or chains (e.g., oligo- or poly(alkylene glycols) as defined herein, urethane, oligourethane or polyurethane moieties as defined herein, and the like, including combinations (e.g., copolymers) of any of the above.

[0197] "Elasticity" refers to the ability of a deformed object to return to its original shape and size when the force that caused the deformation is removed. Elasticity can be determined, for example, by determining the storage modulus, elastic modulus, and / or shear recovery rate of the solidified material. Examples of methods for determining these parameters are described in the Examples section below. Other methods are known in the art and are contemplated.

[0198] The curable elastomeric moiety can be a monofunctional elastomeric moiety containing one curable group or a multifunctional curable moiety containing two or more curable groups.

[0199] The monofunctional curable elastomeric portion according to some embodiments of the present invention may be derived from a vinyl-containing compound represented by Formula I. [ka] wherein at least one of R1 and R2 is and / or includes an elastomeric moiety as described herein.

[0200] The (=CH2) group in Formula I represents a polymerizable group, which according to some embodiments is a UV curable group, such that the elastomerically curable materials and moieties derived therefrom are UV curable materials or moieties.

[0201] For example, R1 is or includes an elastomeric moiety as defined herein, and R2 is, for example, hydrogen, C(1-4) alkyl, C(1-4) alkoxy, or any other substituent, preferably hydrogen or alkyl (e.g., methyl).

[0202] In some embodiments, R1 is a carboxylate and the curable elastomeric moiety is a monofunctional (meth)acrylate. In some of these embodiments, R2 is hydrogen and the curable elastomeric moiety is a monofunctional acrylate. In some of these embodiments, R2 is methyl and the curable elastomeric moiety is a monofunctional methacrylate. Curable moieties where R1 is a carboxylate and R2 is hydrogen or methyl are collectively referred to herein as "(meth)acrylates."

[0203] In some of these embodiments, the carboxylate group -C(=O)-ORa comprises Ra that is or comprises an elastomeric moiety as described herein and is linked to a collagen as described herein. In some embodiments, the Ra elastomeric moiety terminates in a reactive group (e.g., a carboxylate group that reacts with the amine group of a lysine residue of collagen) that is used to conjugate the compound of formula I to the respective group of collagen.

[0204] In some embodiments, R1 is amide and the elastomeric portion is a monofunctional acrylamide. In some of these embodiments, R2 is hydrogen and the curable elastomeric portion is a monofunctional acrylamide. In some of these embodiments, R2 is methyl and the curable elastomeric portion is a monofunctional methacrylamide. Curable elastomeric portions where R1 is amide and R2 is hydrogen or methyl are collectively referred to herein as "(meth)acrylamides."

[0205] (Meth)acrylates and (meth)acrylamides are collectively referred to herein as (meth)acrylic materials.

[0206] In a multifunctional elastomeric segment, two or more polymerizable groups are linked to each other by an elastomeric moiety as described herein, which is also linked to collagen.

[0207] In some embodiments, the multifunctional elastomeric portion can be represented by Formula I described herein, where R1 comprises an elastomeric material terminated with a polymerizable group, as described herein.

[0208] For example, a difunctional elastomeric curing moiety can be represented by formula I*. [ka] wherein E is an elastomeric linking moiety as described herein, and R'2 is as defined herein for R2.

[0209] In some embodiments, multifunctional (eg, difunctional, trifunctional or higher) elastomeric curable materials can be collectively represented by Formula II: [ka] During the ceremony: L represents the point of attachment to the collagen and may be a bond or a linking moiety, such as an alkylene or hydrocarbon chain; R2 and R'2 are as defined herein; B is a tri- or tetrafunctional branching unit as defined herein (depending on the nature of X1); X2 and X3 are each independently selected from absent, an elastomeric moiety as described herein, or an alkyl, a hydrocarbon, an alkylene chain, a cycloalkyl, an aryl, an alkylene glycol, a urethane moiety, and any combination thereof; X1 is absent or selected from alkyl, hydrocarbon, alkylene chain, cycloalkyl, aryl, alkylene glycol, urethane moiety, and elastomeric moiety, each of which is optionally substituted (e.g., terminated) with a meth(acrylate) moiety (OC(=O)CR''2=CH2), and any combination thereof, or X1 is of the formula: [ka] During the ceremony: The curved lines represent the connection points. B' is a branching unit and is the same as or different from B; X'2 and X'3 are each independently as defined herein for X2 and X3; R''2 and R'''2 are as defined herein for R2 and R'2. (provided that at least one of X1, X2 and X3 is or includes an elastomeric moiety as described herein).

[0210] As used herein, the term "branch unit" refers to a multi-radical, preferably an aliphatic or alicyclic group. "Multi-radical" means that the linking moiety has two or more points of attachment such that there is a connection between two or more atoms and / or groups or moieties.

[0211] That is, a branching unit is a chemical moiety that, when attached to a single position, group, or atom of a substance, "branches" a single functional group into two or more functional groups by forming two or more functional groups attached to that single position, group, or atom.

[0212] In some embodiments, the branching unit is derived from a chemical moiety having two, three or more functional groups, hi some embodiments, the branching unit is a branched alkyl or branched linking moiety as described herein.

[0213] Multifunctional elastomeric curable materials having four or more polymerizable groups are also contemplated and can have a structure similar to that shown in formula II, and further can include, for example, a branching unit B that has more branches or an X1 moiety having two (meth)acrylate moieties as defined herein.

[0214] In some embodiments, the elastomeric moiety, such as Ra in Formula I or the moiety shown as E in Formulas I* and II, is or includes an alkyl, which may be linear or branched, preferably 3 or more or 4 or more carbon atoms in length, an alkylene chain, preferably 3 or more or 4 or more carbon atoms in length, an alkylene glycol, as defined herein, an oligo(alkylene glycol) or a poly(alkylene glycol), as defined herein, preferably 4 or more atoms in length, a urethane, oligourethane, or polyurethane, as defined herein, preferably 4 or more carbon atoms in length, and any combination of the above.

[0215] In some of the embodiments described herein, the elastomeric curable material is a (meth)acrylic curable material described herein, and in some embodiments, the elastomeric curable material is an acrylate.

[0216] In some of the embodiments described herein, the elastomeric hardenable moiety is a monofunctional elastomeric hardenable moiety, and in some embodiments, the monofunctional elastomeric hardenable material is represented by Formula I, where R1 is -C(=O)-ORa or -C(=O)-NH-Ra, where Ra is or includes a poly(alkylene glycol) chain as defined herein (e.g., having 4 or more alkylene glycol groups, preferably 6 or more, and preferably 8 or more).

[0217] In some of the embodiments described herein, the elastomeric hardenable moiety is a monofunctional elastomeric hardenable moiety, and in some embodiments, the monofunctional elastomeric hardenable material is represented by Formula I, where R1 is -C(=O)-NH-Ra, where Ra is or includes a poly(alkylene glycol) chain as defined herein (e.g., having 4 or more alkylene glycol groups, preferably 6 or more, and preferably 8 or more alkylene glycol groups).

[0218] In some of the embodiments described herein, the elastomeric hardening moieties are selected such that the elastomeric material from which they are derived, upon solidification (alone), results in a polymeric material with a Tg below 0°C or below -10°C.

[0219] According to some of the embodiments described herein, in each of the curable elastomeric segments, a curable group is at the end of each of the elastic segments, whereby, for example, in Formula I, when Ra is or includes an elastomeric segment, the elastomeric segment is attached to collagen at its other end. It should be noted that alternatively or additionally, curable elastomeric segments having curable groups at positions other than the termini are also contemplated.

[0220] According to some of the embodiments described herein, in at least some or each of the curable elastomeric portions, the curable groups are photocurable or photopolymerizable groups, such as UV-curable groups.

[0221] According to some of the embodiments described herein, in at least some or each of the curable elastomeric portions, the curable group is a (meth)acryl group. In some of these embodiments, the curable group is a (meth)acrylamide, and in some embodiments, a methacrylamide.

[0222] According to some of the embodiments described herein, in at least some or each of the curable elastomeric segments, the elastic segment is or includes a poly(alkylene glycol) segment as described herein, and in some of these embodiments, the poly(alkylene glycol) segment is terminated with a (meth)acryl group (e.g., (meth)acrylamide).

[0223] In at least some or each of the elastomeric segments, the curable group is linked to the elastomeric (e.g., poly(alkylene glycol)) segment by a linking moiety, so that, for example, the (meth)acrylic curable elastomeric segment is represented by Formula A: [ka] During the ceremony: R2 is as defined in any of the embodiments herein; W is -(C=X)-O- or -C=X-NRa; X is O or S; Ra is hydrogen or alkyl; L is a linking moiety, E is an elastomeric moiety, e.g., a poly(alkylene glycol), as defined in any of the embodiments herein; The dashed lines represent points of attachment to collagen (eg, by covalent bonds as described herein).

[0224] In some embodiments, the linking moiety is or comprises an alkylene chain, preferably a short alkylene chain having a length of 10 or less, or 8 or less, or 6 or less, or 4 or less carbon atoms, for example, 1-6, or 1-4 carbon atoms in length.

[0225] In some embodiments, the linking moiety is attached to the elastomeric moiety (e.g., a poly(alkylene glycol) moiety) through a bond, such as an amide bond, a carbamate bond, an ether bond, an ester bond, a thioester bond, a thioamide bond, a thiocarbamate bond, a sulfonamide bond, or the like. In some of these embodiments, the linking moiety is connected to the elastomeric moiety through a carbamate bond.

[0226] According to some of the embodiments described herein, when the elastomeric moiety is or includes a poly(alkylene glycol) moiety, the average molecular weight of a plurality of such moieties is at least 1000 grams / mol, or at least 2000 grams / mol, or at least 3000 grams / mol, or at least 4000 grams / mol, e.g., from about 1000 to about 20,000 grams / mol, from about 2000 to about 20,000 grams / mol, from about 3000 to about 20,000 grams / mol, from about 4000 to about 20,000 grams / mol, from about 1000 to about 15,000 grams / mol, or from about 1000 to about 20,000 grams / mol. The range is about 1000 to about 8000 g / mol, about 2000 to about 8000 g / mol, about 3000 to about 8000 g / mol, about 4000 to about 15000 g / mol, about 1000 to about 10000 g / mol, about 2000 to about 10000 g / mol, about 3000 to about 10000 g / mol, about 4000 to about 10000 g / mol, or about 1000 to about 8000 g / mol, about 2000 to about 8000 g / mol, about 3000 to about 8000 g / mol, or about 4000 to about 8000 g / mol (including intermediate values ​​and partial ranges therebetween).

[0227] Each of the curable elastomeric moieties described herein in any of the embodiments can be linked to collagen by a covalent bond between a functional (reactive) group of the curable elastomeric moiety and a functional group of collagen, preferably a functional group at the collagen termini and / or a functional group of an amino acid side chain. The curable elastomeric moieties can be linked to collagen by the same or different bonds.

[0228] According to some of the embodiments described herein, at least some or all of the elastomeric portion is covalently bonded to lysine residues of collagen.

[0229] According to some of the embodiments described herein, at least a portion or all of the elastomeric portion is covalently bonded to the collagen via a carbamate bond.

[0230] According to some of the embodiments described herein, at least some or all of the elastomeric portion is covalently bonded to lysine residues of collagen via bonds such as amide bonds, carbamate bonds, thioamide bonds, thiocarbamate bonds, sulfonamide bonds, hydrazine bonds, hydrazine bonds, and the like.

[0231] According to some of the embodiments described herein, at least some or all of the elastomeric portion is covalently attached to lysine residues of collagen via carbamate bonds.

[0232] According to some of the embodiments described herein, at least 1%, e.g., 1-20%, or 1-10%, or at least 2%, e.g., 2-20%, or 2-10%, of the lysine residues in the collagen have a curable elastomeric moiety covalently attached (e.g., via a carbamate bond).

[0233] An exemplary conjugate according to this embodiment is shown in FIG. 1 and is also referred to herein as "CPM."

[0234] According to some of the embodiments described herein, the collagen to which the curable elastomeric portion is attached is as described herein in any of the embodiments.

[0235] According to some of the embodiments described herein, the collagen is human type I collagen as described herein.

[0236] According to some of the embodiments described herein, the collagen is a recombinant collagen as described herein.

[0237] According to some of the embodiments described herein, the collagen is a recombinant collagen derived from a plant as described herein.

[0238] According to some of the embodiments described herein, the collagen is a plant-derived recombinant human type I collagen as described herein, such as tobacco-derived collagen.

[0239] According to some of the embodiments described herein, the collagen to which the elastomeric curable groups are attached has multiple curable groups, e.g., photocurable groups, other than the curable elastomeric moiety, such that in some embodiments, the conjugate comprises a curable collagen as described herein in any of the embodiments, having attached thereto a curable elastomeric moiety as described herein in any of the embodiments, and any combination thereof.

[0240] The exemplary sclerotic collagen according to this embodiment is also referred to herein as "CMR."

[0241] An exemplary conjugate comprising a curable collagen as described herein in any of the embodiments having a curable elastomeric moiety attached thereto is also referred to herein as a "CPMR."

[0242] According to one aspect of some embodiments of the present invention there is provided a process for preparing the conjugates described herein.

[0243] Generally, this process is carried out by coupling an elastomeric moiety, terminated at one end with a curable group as described herein and at the other end with a first reactive moiety, to collagen as described herein, where contact is carried out under conditions that allow reaction between the first reactive moiety and a chemically compatible portion of the collagen.

[0244] According to some embodiments, the process further comprises, prior to coupling, preparing an elastomeric portion terminated at one end with a curable group as described herein and at the other end with a first reactive moiety.

[0245] Exemplary synthetic routes are described in further detail in the Examples section below.

[0246] According to some of the embodiments described herein, there is provided an elastomeric settable collagen that is a conjugate of a settable collagen described herein in any of the embodiments and any combination thereof and a plurality of elastic moieties attached to the settable collagen. In some of these embodiments, the elastic moieties do not have a settable group. The elastic moieties may be the same or different, and each may independently be an elastic moiety described herein in any of the embodiments. In exemplary embodiments, at least a portion of the elastic moieties comprise a poly(alkylene glycol) moiety described herein in any of the embodiments and any combination thereof. In some of these exemplary embodiments, at least a portion of the poly(alkylene glycol) moieties are "capped", i.e., terminated with a group other than hydroxy, such as alkoxy (e.g., methoxy). In exemplary embodiments, the conjugate comprises a plurality of poly(ethylene glycol) moieties, each of which has an average molecular weight of about 5,000 or 6,000 grams / mol, and each of which terminates with a methoxy group. An exemplary conjugate according to these embodiments is described in Example 6 of the Examples section below, and is also referred to as "PCMR".

[0247] Curable formulation: According to one aspect of some embodiments of the present invention, there is provided a formulation (or composition) comprising the conjugate described herein, also referred to herein as a hardenable composition or hardenable formulation. According to some embodiments, the hardenable composition can be used in additive manufacturing (e.g., bioprinting) of 3D objects described herein. According to some embodiments, the composition can be used or is for use in the preparation of or as one or more modeling material formulations for additive manufacturing processes (e.g., bioprinting). Additive manufacturing is the additive manufacturing of a three-dimensional object comprising at least a portion thereof a collagen material described herein.

[0248] According to some of the present embodiments, a composition comprising the conjugates described herein is also referred to herein as a bio-ink composition or bio-ink formulation, or simply bio-ink.

[0249] According to some of the embodiments described herein, the curable formulation further comprises a carrier, which in some embodiments is an aqueous carrier.

[0250] The aqueous carrier can be water, a buffer solution characterized by a pH range of about 2 to about 10, or about 2 to about 9, or about 3 to about 9, or about 3 to about 8, a basic aqueous solution, or an acidic aqueous solution.

[0251] 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.

[0252] 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.

[0253] In some embodiments, the aqueous carrier comprises a phosphate salt, such as monobasic sodium phosphate (NaH2PO4) and / or dibasic sodium phosphate (sodium hydrogen phosphate, Na2HPO4). In some embodiments, the total concentration of the phosphate salt in the formulation is about 0.1 M.

[0254] In some embodiments, the aqueous carrier comprises NaCl or any other physiologically acceptable salt.

[0255] 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.

[0256] Phosphate buffered saline (PBS) can be commercially available PBS (eg, DPBS) or a custom buffer characterized by the desired pH and / or osmolality.

[0257] 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.

[0258] Any other buffer may be used in the context of this embodiment.

[0259] In some of the embodiments described herein, the aqueous carrier comprises an acid.

[0260] 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).

[0261] The acid can be an inorganic acid (eg, HCl) or an organic acid (preferably water soluble at the concentrations described above (eg, acetic acid)).

[0262] 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).

[0263] 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.

[0264] According to certain of the embodiments described herein, the hardenable formulation has a pH in the range of from about 2 to about 9, or from about 3 to about 9, or from about 3 to about 8.5, or from about 3 to about 8, or from about 3.5 to about 9, or from about 3.5 to about 8.5, or from about 3.5 to about 8, or from about 4 to about 8.5, or from about 4 to about 8, or from about 4.5 to about 8.5, or from about 4.5 to about 8, or from about 5 to about 8.5, or from about 5 to about 8, or from about 5.5 to about 8.5, or from about 5.5 to about 8, or from about 6 to about 8 (including any intermediate or subrange therebetween).

[0265] In some of the embodiments described herein, the concentration of the conjugate described in any of the embodiments and any combination thereof herein in the curable 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 40 mg / mL, or 1 mg / mL to 30 mg / mL, or 1 mg / mL to 20 mg / mL, or 0.5 mg / mL to 10 mg / mL, or 1 mg / mL to 10 mg / mL (including any intermediate values ​​and subranges therebetween).

[0266] The concentration of the conjugate in the curable formulation can affect the rheological properties of the formulation and the resulting solidified material upon curing (e.g., exposure to curing conditions such as irradiation), and can be manipulated depending on the AM method and conditions used and the desired properties of the final object or portion thereof.

[0267] According to some of the embodiments described herein, the curable formulation is characterized by shear thinning behavior (eg, at room temperature, such as 20-25° C.) and is a shear thinning composition.

[0268] The term "shear thinning" describes the property of a fluid material as reflected by a decrease in viscosity (increased fluidity) upon application of a shear force (under shear strain) at a specified temperature, as determined using a rheometer as described in the Examples section below.

[0269] In some of the present embodiments, the shear thinning material experiences a significant (e.g., at least 100%) decrease in its shear modulus when the shear strain is increased from about 1% to greater than 50%, and thus the shear thinning material exhibits a shear dependent viscosity profile.

[0270] According to some of the embodiments described herein, the curable formulations are characterized by a fast rate of recovery in response to changes in applied shear force (fast shear recovery).

[0271] According to some of the embodiments described herein, the curable formulation is characterized by a change of 6% or less, or 10% or less, after 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, or even 10 minutes of shear rest (zero shear force).

[0272] According to some of the embodiments described herein, the curable formulation is characterized by having a viscosity that recovers at least 80%, or at least 85%, or at least 90%, or at least 92% when the shear rate is increased from about 0 (1 / sec) or 1 (1 / sec) to greater than 50 (1 / sec) over a period of at least 1 minute (e.g., from about 60 seconds to about 120 seconds, e.g., about 100 seconds).

[0273] According to some of the embodiments described herein, the curable formulation is characterized by a viscosity of 200 centipoise or less, or 250 centipoise or less, at a shear rate of 10 (1 / sec) and room temperature, as described herein, using a rheometer as described in the Examples section below.

[0274] According to some of the embodiments described herein, the settable formulation is characterized by any of the above-mentioned viscosity / rheological behaviors when the concentration of the settable collagen is at least 2 mg / mL, or at least 3 mg / mL, or at least 4 mg / mL, or from about 2 mg / mL to about 10 mg / mL, or in the range of about 2 mg / mL to about 5 mg / mL (including any intermediate values ​​and subranges therebetween).

[0275] According to some of the embodiments described herein, the concentration of the conjugate in the bioink composition can be in the range of about 0.1 to about 20, or 0.1 to about 10, or about 1 to about 20, or about 5 to about 20, or about 8 to about 20, or about 10 to about 20, or about 5 to about 15, or about 8 to about 15, or about 8 to about 12, or about 10 to about 15, or about 1 to about 10, or about 2 to about 10, or about 2 to about 5, or about 3 to about 10, or about 3 to about 5, or about 2 to about 8, or about 2 to about 6, or about 4 to about 10, or about 4 to about 8, or about 4 to about 6 mg / mL, or about 5 to about 10, or about 6 to about 10, or about 8 to about 10 mg / mL (including intermediate values ​​and subranges therebetween).

[0276] According to some of the embodiments described herein, the curable formulation, upon solidification, forms a hydrogel material by crosslinking of the conjugates within the aqueous carrier.

[0277] 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).

[0278] 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.

[0279] Hydrogels can take physical forms ranging from soft, brittle and weak to hard, elastic and tough. Soft hydrogels can be 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.

[0280] 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.

[0281] According to some of the embodiments described herein, the bio-ink composition is characterized by a storage modulus (G') of 25,000 Pa or less when solidified.

[0282] According to some of the embodiments described herein, the bio-ink composition, when solidified, is characterized by a storage modulus (G') that is at least 5,000 Pa, or at least 6,000 Pa, or at least 8,000 Pa, or at least 10,000 Pa lower than the storage modulus of a comparative bio-ink composition comprising a settable collagen that does not have a settable elastic moiety. According to some of these embodiments, the comparative bio-ink composition comprises the same amount of settable collagen as the conjugate of the present embodiments, and the same carriers and additives.

[0283] According to some of the embodiments described herein, the bioink composition, when solidified, has a storage modulus (G') of about 100 Pa to about 50,000 Pa, or about 1,000 Pa to about 50,000 Pa, or about 100 Pa to about 40,000 Pa, or about 1,000 Pa to about 40,000 Pa, or about 100 Pa to about 30,000 Pa, or about 1,000 Pa to about 30,000 Pa, or about 100 Pa to about 25,000 Pa, or about 1,0 The pressure is characterized by being in the range of about 00 Pa to about 25,000 Pa, or about 1,000 Pa to about 20,000 Pa, or about 100 Pa to about 20,000 Pa, or about 1,000 Pa to about 20,000 Pa, or about 5,000 Pa to about 30,000 Pa, or about 5,000 Pa to about 25,000 Pa, or about 10,000 Pa to about 30,000 Pa, or about 10,000 Pa to about 25,000 Pa (including intermediate values ​​and partial ranges therebetween).

[0284] According to some embodiments of the present invention, hydrogels 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 intermingling. Such polymeric fibrous elements may be woven (e.g., as in a mesh structure) or nonwoven, and in some embodiments may function as reinforcing materials for the hydrogel's fibrous network. Non-limiting examples of such polymers include polycaprolactone, gelatin, crosslinked gelatin (e.g., formed with gelatin methacrylate), alginate, crosslinked alginate (e.g., formed with alginate methacrylate), chitosan, crosslinked chitosan (e.g., formed with chitosan methacrylate), glycol chitosan, crosslinked glycol chitosan (e.g., formed with glycol chitosan methacrylate), hyaluronic acid (HA), crosslinked hyaluronic acid (e.g., formed with HA methacrylate), and other crosslinked or non-crosslinked natural or synthetic polymer chains. Alternatively or additionally, such polymers are chemically bound to the main crosslinked network of the hydrogel, for example by acting as crosslinkers or by forming part of the three-dimensional network of the hydrogel.

[0285] 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.

[0286] According to some of the embodiments described herein, the hardenable 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.

[0287] According to some of the embodiments described herein, the print medium (a build material described herein, which may include one, two, or more modeling material formulations, e.g., one or more bio-ink formulations) includes one or more additional materials, e.g., one or more additional curable materials, one or more non-curable materials, and / or one or more biological components.

[0288] According to some of the embodiments described herein, the additional material is included in the conjugate-containing curable formulation (referred to herein as a bio-ink composition) or one or more other modeling material formulations.

[0289] Additional curable materials that may be included in the conjugate-containing curable formulations of this embodiment or one or more other modeling material formulations may be any curable material as defined herein, and are preferably biocompatible materials.

[0290] In some embodiments, the additional 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 or hydrogel-forming materials.

[0291] 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.

[0292] According to embodiments of the present invention, the hydrogel may be of biological origin or synthetically prepared.

[0293] 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.

[0294] 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 cross-linking (e.g., curable) groups or can be used in combination with a compatible cross-linking agent.

[0295] 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.

[0296] Hydrogel precursors (hydrogel-forming materials) that form such polymer chains (including any combination thereof) are contemplated.

[0297] 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.

[0298] 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). n Examples 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.

[0299] 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.

[0300] 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 poly(ethylene glycol): PEG, gelatin methacrylate: GelMA, poly(propylene oxide): PPO, poly(ethylene oxide): PEO, PEG( Poly(ethylene glycol) diacrylate, polyglutamic acid, PLGA / PLLA, poly(dimethylsiloxane); nanocellulose; Pluronic F127, short dipeptides (FF), Fmoc-peptide based hydrogels such as 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).

[0301] 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, genipin, or chitosan that can be crosslinked with chemical agents such as sodium tripolyphosphate (TPP).

[0302] According to some of the embodiments described herein, the additional curable material has one or more curable groups that polymerize and / or crosslink under the same conditions as the curable elastomeric moieties in the conjugates of the present embodiments. In some of these embodiments, the additional curable material has photocurable (e.g., UV-curable) groups, such as acrylic groups as described herein.

[0303] According to some of the embodiments described herein, the additional curable material is or includes a poly(alkylene glycol) such as poly(ethylene glycol) having one or more photocurable groups, e.g., one or more acrylic groups as described herein. In some of these embodiments, the additional curable material can be, for example, a poly(alkylene glycol) (meth)acrylate, such as poly(ethylene glycol) (meth)acrylate, and / or a poly(alkylene glycol) di(meth)acrylate, such as poly(ethylene glycol) di(meth)acrylate, and / or a copolymer including the above, e.g., polycaprolactone (meth)acrylate and / or di(meth)acrylate / poly(ethylene glycol), poly(lactic acid) (meth)acrylate and / or di(meth)acrylate / poly(ethylene glycol), poly(lactic acid-co-glycolic acid) (meth)acrylate and / or di(meth)acrylate / poly(ethylene glycol), including any combination of the above.

[0304] According to some of the embodiments described herein, the additional material is or includes a poly(alkylene glycol) terminated with at least one (meth)acrylic group, i.e., the additional material is a polymer or copolymer of a poly(alkylene glycol) terminated with at least one (meth)acrylic group.

[0305] According to some of the embodiments described herein, the poly(ethylene glycol) or copolymers thereof having curable groups have an average molecular weight of at least 500 grams / mol or at least 700 grams / mol. In some embodiments, the average molecular weight is less than 4,000 grams / mol, or less than 3,000 grams / mol, or less than 2,000 grams / mol, or less than 1,000 grams / mol. In some embodiments, the average molecular weight is in the range of 500-30,000, or 500-20,000, or 50-10,000, or 500-5,000, or 500-4,000, or 500-3,000, or 500-2,000 grams / mol (including any intermediate values ​​or subranges therebetween). In some embodiments, the average molecular weight is in the range of 3,000 to about 30,000, or 3,000 to about 20,000, or 3,000 to about 10,000 grams / mol (including any intermediate or subrange therebetween).

[0306] According to some of the embodiments described herein, the concentration of the additional hardenable material is in the range of 1 to 30, or 1 to 20, or 1 to 10, or 5 to 20, or 5 to 15, or 10 to 20, or 10 to 30 weight percent of the total weight of the composition, including any intermediate values ​​and subranges therebetween.

[0307] As described herein, the conjugate-containing curable formulations according to any of the embodiments can be used as one or more modeling material formulations for additive manufacturing processes (e.g., bioprinting), as described in more detail below.

[0308] Where a conjugate-containing curable formulation is described as including components other than the conjugate and carrier described herein, the additional components can be included in the same modeling material formulation or in a different modeling material formulation used for additive manufacturing in combination with the conjugate-containing curable formulation.

[0309] For example, where an additional curable material other than the conjugate of the present embodiment is described, the additional curable material can be included in the same modeling formulation that includes the conjugate and / or in a different modeling material formulation.

[0310] In general, the components described herein for inclusion in the bio-ink composition can be included in the same modeling formulation or can be included together in the bioprinting medium, build material, and can be split into two or more modeling material formulations as needed, so long as they meet the requirements for additive manufacturing.

[0311] According to some of the embodiments described herein, the print medium (build material) in general or the conjugate-containing hardenable formulation in particular further comprises biological components or materials other than collagen (also collectively referred to herein as biological materials).

[0312] Biological components or materials that can be included in one or more of the hardenable (e.g., 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.

[0313] 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.

[0314] According to one embodiment of this aspect of the invention, the cells are of mammalian origin.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] A non-hardenable material other than a biological material as described herein that may be included in one or more of the hardenable (e.g., modeling material) formulations described herein may be a material that imparts a particular property to the formulation or to the solidified formulation or material and to a portion of the object formed therefrom. Such a property may be a physical property (e.g., optical property such as transparency or opacity, color, spectral property, heat resistance, electrical property, etc.), or a mechanical or rheological property such as viscosity, elasticity, storage modulus, loss modulus, stiffness, hardness, etc. Alternatively or additionally, the non-hardenable material may provide a biological function, e.g., a therapeutically active agent.

[0319] Examples of non-curable materials include thixotropic agents, reinforcing agents, toughening agents, fillers, colorants, pigments, color materials (such as those described herein), and the like.

[0320] An example of a non-curable material is titanium dioxide.

[0321] An example of a non-hardening material is oxidized cellulose.

[0322] According to some of the embodiments described herein, one or more of the hardenable (eg, modeling material) compositions includes hyaluronic acid.

[0323] According to some of the embodiments described herein, one or more of the hardenable (eg, modeling material) formulations comprises hyaluronic acid having hardenable groups as defined herein.

[0324] According to some of the embodiments described herein, one or more of the hardenable (e.g., modeling material) compositions include one or more biological components or materials (e.g., including, but not limited to, cells, growth factors, peptides, heparan sulfate, and fibronectin).

[0325] According to some of the embodiments described herein, one or more of the hardenable (e.g., modeling material) compositions include one or more agents that modify the mechanical properties of the compositions and / or objects described herein (e.g., but are not limited to, alginates, hyaluronic acid, fibrinogen, elastin, peptides, and thixotropic agents (e.g., crystalline nanocellulose (CNC)), oxidized cellulose, titanium dioxide, clay minerals, and carbon nanotubes).

[0326] In some of the embodiments described herein, the conjugate-containing curable formulation further comprises one or more additional curable materials as described herein in any of the embodiments.

[0327] In some of these embodiments, the weight ratio of the conjugate to the additional curable material in the formulation is in the range of 20:1 to 1:2, or 20:1 to 1:1, or 10:1 to 1:1, or 20:1 to 5:1, or 15:1 to 5:1 (including any intermediate or subrange therebetween).

[0328] In some of the embodiments described herein, the conjugate-containing curable formulation further comprises a thixotropic agent as defined herein.

[0329] 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.

[0330] In some of the embodiments described herein, the conjugate-containing curable formulation further comprises a gel former, such as a hydrogel former described herein.

[0331] In some of the embodiments described herein, the conjugate-containing curable formulation further comprises a biological component or material as described herein.

[0332] In some of the embodiments described herein, the conjugate-containing curable formulation further comprises one or more curable or non-curable materials as described in any of the embodiments herein.

[0333] According to some of the embodiments described herein, the conjugate-containing curable formulation further comprises one or more biological components, such as, but not limited to, hyaluronic acid (including curable HA), cells, growth factors, peptides, heparan sulfate, and / or fibronectin.

[0334] According to some of the embodiments described herein, the conjugate-containing hardenable 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)).

[0335] In some embodiments using two or more modeling material formulations, the two or more formulations are conjugate-containing curable formulations 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 a conjugate described herein, and another formulation can include a conjugate and a biological material described herein. For example, one formulation can include a conjugate described herein, and another formulation can include a conjugate described herein and an additional curable material described herein. For example, one formulation can include a conjugate described herein and an additional curable material, and another formulation can include a conjugate described herein and an additional curable material described herein. For example, one formulation can include a conjugate described herein and an additional curable material, and another formulation can include a conjugate described herein and an additional curable material described herein. For example, one formulation can include a conjugate described herein and an additional curable material, and another formulation can include a curable conjugate described herein and a non-curable material (e.g., a biological material or component) described herein. Any other combinations are contemplated.

[0336] In some of the embodiments described herein, all of the hardenable materials in the build material are cured under the same curing conditions, hi some embodiments, all of the hardenable materials or hardenable groups are photocurable.

[0337] In some of the embodiments described herein, the formulations comprising the hardenable materials described herein further comprise an agent that promotes the hardening or solidification of the hardenable material upon exposure to curing conditions.

[0338] 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).

[0339] Photoinitiator: When the curable material is a photocurable material (e.g., a UV-curable material, such as a curable material having one or more (meth)acrylic groups), the agent is a photoinitiator. The photoinitiator is selected depending on the curing mechanism (e.g., free radical, cationic, etc.).

[0340] A free radical photoinitiator can be any compound that generates free radicals upon exposure 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-trimethylbenzolidiphenylphosphine oxide (TMPO) (e.g., lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 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-amino ketones and bisacylphosphine oxides (BAPO).

[0341] Examples of photoinitiators include, but are not limited to, the Irgacure® family, riboflavin, rose bengal, and the like.

[0342] 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.

[0343] 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.

[0344] 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).

[0345] According to some of the embodiments described herein, the photoinitiator is a free radical photoinitiator as described herein, such as an acylphosphine oxide type photoinitiator.

[0346] According to some of the embodiments described herein, the amount of photoinitiator in the formulation ranges from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 3, or from about 0.1 to about 2, or from about 0.1 to about 1 weight percent, including any intermediate values ​​and subranges therebetween.

[0347] The inventors have surprisingly found that there is a direct correlation between the amount of photoinitiator and the viscosity of a formulation comprising a curable collagen (e.g., designated as CMR or CPM) as described herein and the photoinitiator (see FIG. 8). More specifically, the inventors have shown that by manipulating the amount of an exemplary acylphosphine oxide-type photoinitiator, the viscosity of the formulation can be controlled and thus tailored to the viscosity desired for a particular AM process. Without being bound to any particular theory, it is hypothesized that the effect on the viscosity of the formulation is the result of a unique interaction between the photoinitiator and the curable collagen.

[0348] According to an aspect of some embodiments of the present invention, there is provided a method for additive manufacturing of a three-dimensional object comprising at least a portion of a collagen-based material, performed as described herein, and further comprising, prior to dispensing the collagen-containing hardenable formulation, selecting an amount of photoinitiator that results in a hardenable collagen-containing formulation exhibiting a viscosity suitable for a selected additive manufacturing application, preparing the collagen-containing hardenable formulation with the selected amount of photoinitiator, and dispensing the formulation described herein.

[0349] According to one aspect of some embodiments of the present invention, there is provided a method for preparing a formulation usable for additive manufacturing of a three-dimensional object comprising at least in part a collagen-based material, the method comprising the steps of: Selecting additive manufacturing technologies; Determining a suitable viscosity for additive manufacturing techniques; Determining the amount of photoinitiator that will give the desired viscosity; and Methods are provided that include mixing the determined amount of a photoinitiator with a selected hardenable collagen (e.g., as described herein in any of the embodiments and any combination thereof), and optionally with other ingredients included in the hardenable formulations described herein, to prepare a formulation.

[0350] Throughout this specification, particularly in relation to additive manufacturing, the terms "method" and "process" are used interchangeably.

[0351] The method of the present embodiment can be performed using a look-up table that defines the appropriate viscosity for each of the various AM techniques and defines the amount of photoinitiator to obtain the desired viscosity, or is based on published knowledge of the appropriate viscosity values ​​for each AM technique that already defines the amount of photoinitiator required to obtain the appropriate viscosity for each technique.

[0352] Thus, the method of preparing a curable formulation can be carried out by mixing an amount of photoinitiator with a selected curable collagen (e.g., as described herein in any of the embodiments and any combination thereof), and optionally with other ingredients included in the curable formulation described herein, the amount being determined based on the look-up table described above.

[0353] Preparation of the formulation can be done manually or automatically, after manual or automatic calculation of the amount of photoinitiator.

[0354] According to some of the embodiments described herein, the photoinitiator is an acylphosphine oxide type photoinitiator, such as 2,4,6-trimethylbenzolidiphenylphosphine oxide (TMPO) or a salt thereof (e.g., lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2,4,6-trimethylbenzoylethoxyphenylphosphine oxide (TEPO), and bisacylphosphine oxide (BAPO).

[0355] According to some embodiments of this aspect of the invention, the collagen is human type I collagen as described herein, preferably recombinant human type I collagen as described in any of the embodiments herein.

[0356] Table 1 below is an example of a desirable look-up table as described above, showing desirable viscosity values ​​of modeling material formulations for a representative AM process, and the respective amounts of photoinitiators to be included in the formulation (e.g., including the curable collagen described herein for CMR).

[0357] [Table 1]

[0358] 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.

[0359] 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 can 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.

[0360] 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."

[0361] The build material can include one, two, or more modeling material formulations, where at least one of the modeling material formulations includes a conjugate described herein and / or is a curable formulation described herein in any of the embodiments and any combination thereof.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] When AM is performed by 3D inkjet printing, an uncured build material as defined herein is dispensed from a dispensing head having 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 of which may be configured to dispense different build materials (e.g., different modeling material formulations (each of which may include different biological components, or different curable materials, or different concentrations of curable materials) and / or different support material formulations). Thus, different target locations may be occupied with different build materials (e.g., modeling formulations and / or support formulations as defined herein).

[0366] 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).

[0367] 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.

[0368] 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).

[0369] 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.

[0370] Depending on the additive manufacturing method used and the configuration selected, dispensing can be done in the form of droplets or a continuous stream.

[0371] 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.

[0372] 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.

[0373] Once the uncured build material has been dispensed onto the receiving medium according to the 3D data, the method then optionally and preferably solidifies the dispensed formulation. 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.

[0374] 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).

[0375] 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.

[0376] 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).

[0377] 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.

[0378] "Curing energy" typically includes the application of radiation or the application of heat.

[0379] 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.

[0380] Curable materials or systems that harden upon exposure to radiation are referred to interchangeably herein as "photopolymerizable" or "photoactivatable" or "photohardenable."

[0381] 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.

[0382] 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.

[0383] 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).

[0384] Curable materials or systems that harden upon exposure to heat are referred to herein as "thermosetting" or "thermoactivatable" or "thermopolymerizable."

[0385] 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.

[0386] 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.

[0387] 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.

[0388] In some embodiments, the post-treatment is by exposure to heat or radiation, as described in any of the embodiments herein.

[0389] In some embodiments, it is contemplated that different formulations are dispensed from different dispensing heads to manufacture an object, in particular providing the ability to select a formulation from a predefined number of formulations and to define a desired combination of the selected formulation and its properties.

[0390] 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.

[0391] The present embodiment thus enables a wide range of material combinations to be deposited, enabling the fabrication of objects comprised of various combinations of multiple modeling material formulations, with different modeling material formulations disposed in different parts of the object according to the desired properties characterizing each part of the object.

[0392] 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.

[0393] 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.

[0394] 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.

[0395] 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.

[0396] 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.

[0397] According to the present embodiment, the additive manufacturing methods described herein are for bioprinting biological objects.

[0398] 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.

[0399] 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.

[0400] 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., a conjugate described herein), also referred to herein and in the art as a "bio-ink" or "bio-ink formulation" or "bio-ink composition."

[0401] 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.

[0402] 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.

[0403] In some of the embodiments described herein, the build material (e.g., print medium) comprises a modeling material formulation (e.g., a bio-ink composition described herein) 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 component.

[0404] 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 composition.

[0405] 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.

[0406] 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.

[0407] According to this embodiment, additive manufacturing is additive manufacturing of a three-dimensional object characterized at least in part 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, and for at least a portion of the plurality of layers, the dispensing is dispensing of one or more modeling material formulations comprising a bio-ink composition as described herein in any of the embodiments and any combination thereof.

[0408] According to some of the embodiments described herein, the process further comprises exposing at least a portion of the dispensed layer to suitable curing conditions to solidify the bio-ink composition. In some of these embodiments, the curing conditions comprise curing energy, such as light energy (irradiation, illumination).

[0409] According to some of the embodiments described herein, for at least a portion of the multiple layers, 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 comprised of the formulation.

[0410] According to some of the embodiments described herein, the dispensing for at least a portion of the plurality of layers is a further dispensing of a modeling material formulation that includes a biological material other than collagen as described herein.

[0411] According to some of the embodiments described herein, the temperature at which dispensing is performed ranges from −10 to 50° C., or −4 to 50° C., or −4 to 37° C. In some embodiments, the temperature is at least 10° C., or at least 20° C., or 37° C.

[0412] 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.

[0413] 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.

[0414] 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).

[0415] 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).

[0416] 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.

[0417] 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.

[0418] Below, examples of AM bioprinting methods that can be used in embodiments of the present invention are described.

[0419] 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.

[0420] 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.

[0421] In general, bioprinting can be performed using any of the known techniques for additive manufacturing. Some exemplary additive manufacturing techniques are listed below, although any other techniques are contemplated.

[0422] 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.

[0423] 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.

[0424] 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.

[0425] 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 to create models, prototypes, patterns, and production parts in various industries, including bioprinting. DLP differs from laser-based SLA in that it uses the projection of ultraviolet (UV) light (or visible light) from a digital projector to flash a single image of a layer at a time over the entire uncured material. One of the main components of DLP is the digital micromirror device (DMD) chip. It is typically composed of an array of reflective aluminum micromirrors that redirect the incoming light from a UV source and project an image of the designed pattern. To obtain high-resolution structures, parameters such as the curing time, layer thickness, and intensity of the UV light for each layer must be adjusted, for example by controlling the concentration and type of curable material and photoinitiator.

[0426] 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.

[0427] 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.

[0428] Electrospinning: Electrospinning is a fiber-producing technique that uses electrical forces to draw electrically charged threads of a polymer solution or melt.

[0429] According to some of the embodiments described herein, the additive manufacturing (bioprinting) is or includes digital light processing (DLP) as described herein.

[0430] 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.

[0431] As used throughout this specification, the term "object" refers to the entire object or a portion thereof.

[0432] In the context of this embodiment, the object comprises at least a portion of a collagen-based material.

[0433] 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.

[0434] In some of the embodiments described herein, the collagen-based material comprises a scaffold, e.g., a hydrogel scaffold formed of a three-dimensional fibrous network that includes a collagen described herein (e.g., recombinant human collagen).

[0435] In some of the embodiments described herein, the collagen-based materials include polymerized and / or cross-linked (e.g., recombinant human) collagen in which multiple monomeric and / or fibrous collagen units are linked together to form a three-dimensional network.

[0436] 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.

[0437] 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.

[0438] 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.

[0439] In some embodiments, the object is usable or intended for construction of a prosthetic organ or tissue.

[0440] 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.

[0441] 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.

[0442] 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.

[0443] The films can be used to construct biomedical devices, such as collagen membranes for hemodialysis.

[0444] According to some embodiments, the film or scaffold may be used in cell culture.

[0445] 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.

[0446] In some embodiments, the films or scaffolds may be used in the wound healing process.

[0447] 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.

[0448] 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.

[0449] kit: According to an aspect of some embodiments of the present invention there is provided a kit comprising a conjugate as described herein in any of the embodiments.

[0450] According to some embodiments, the kit comprises a curable formulation (e.g., a bio-ink composition) comprising a conjugate described in any of the embodiments herein.

[0451] According to some embodiments, the curable formulation comprises the conjugate in lyophilized form.

[0452] According to some embodiments, the curable formulation comprises a conjugate described herein and an aqueous solution or carrier.

[0453] 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.

[0454] 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 conjugate or composition and the aqueous carrier are packaged separately within the kit.

[0455] Alternatively, the kit comprises instructions for mixing the conjugate or composition with an aqueous carrier to prepare a modeling material formulation as described herein.

[0456] The kit may further include other components that can be included in the bio-ink compositions or modeling material formulations described herein in any of the embodiments and any combination thereof.

[0457] The kit may further comprise instructions on how to use the conjugate or bio-ink composition or formulation in the additive manufacturing processes described herein.

[0458] Pigment substance: According to some of the embodiments described herein, the bio-ink compositions or curable (e.g., modeling material) formulations described herein can include a dye substance capable of absorbing light in a desired wavelength range. Digital light processing bioprinting typically requires the addition of absorbing dye substances to improve resolution and obtain defined porosity and channels in the scaffold.

[0459] According to some of the embodiments described herein, the dye material is such that it can absorb light at wavelengths from 300 nm to 800 nm, or from 300 nm to 600 nm, or from 300 nm to 500 nm, or from 300 nm to 450 nm, or from 350 nm to 450 nm, or preferably from 365 nm to 405 nm.

[0460] The colorant materials described herein are also referred to as light absorbers or light blockers throughout this specification.

[0461] Exemplary color substances suitable for use in connection with these embodiments include food coloring, tartrazine, Sunset Yellow FCF (Yellow No. 6), Brilliant Blue FCF (FD&C Blue No. 1), Indigo Carmine (FD&C Blue No. 2), Fast Green FCF (FD&C Green No. 3), anthocyanins, anthocyanidins, erythrosine (FD&C Red No. 3), Allura Red AC (FD&C Red No. 40), riboflavin (vitamin B2, E101, E101a, E106), ascorbic acid (vitamin C), quinoline yellow WS, carmoisine (azorubine), Ponceau 4R (E124), patent blue V (E13), and the like. 1), Green S (E142), Yellow 2G (E107), Orange GGN (E111), Red 2G (E128), caramel color, phenol red, methyl orange, 4-nitrophenol, and NADH disodium salt, curcumin (E100), turmeric, alpha-carotene, beta-carotene, canthaxanthin (ketocarotenoid), cochineal extract, paprika, saffron, ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), citrus red 2, annatto extract, avobenzone, 2,5-bis(5-tert-butyl-benzoxazol-2-yl)thiophene (Benetex OB+), 4,4'-bis(2-sulfonatostyryl)biphenyl disodium (Benetex OB-M1), benzenepropanoic acid (BLS 99-2), 2,3,6,7-tetrahydro-9-methyl-1H,5H-quinolizino (9,1-gh) coumarin (coumarin 102), maltose yellow, morin hydrate, nitrofurazone, 2-nitrophenyl phenyl sulfide (NPS), 5,12-naphthacenequinone (NTAQ), octocrylene, phenazine, 1,4-bis(2-(5-phenyloxazolyl))benzene (POPOP), quinoline yellow, 3,3',4',5,6-pentahydroxyflavone (quercetin), salicylaldehyde, Sudan I, triamterene, UV386A, 1-phenylazo-2-naphthol (Sudan I), 1-(2,4-Dimethylphenylazo)-2-naphthol (Sudan II), 1-(4-(phenyldiazenyl)phenyl)azonaphthalen-2-ol (Sudan III), 1-[{2-methyl-4-[(2-methylphenyl)diazenyl]phenyl}diazenyl]naphthalen-2-ol (Sudan IV), 2,5-bis(5-tert-butyl-benzoxazol-2-yl)thiophene, fluorescein, poly(3-hexylthiophene-2,5-diyl), oligothiophenes, triphenylamines, diketopyrrolopyrrole derivatives, 2,5-dihydro-3,6-di-2-thienyl-pyrrolo[3,4-c]pyrrole-1,4-dione, boron dipyrromethene derivatives, 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroborane These include, but are not limited to, diazaindacene, 2,2'-(2,5-thiophenediyl)bis(5-tert-butylbenzoxazole), (±)-α-tocopherol, 2-phenyl-2H-benzotriazole derivatives, 2,2-dimethyl-1,3-dihydroperimidin-6-yl)-(4-phenylazo-1-naphthyl)diazene (Sudan Black B), 1-(2-methoxyphenylazo)-2-naphthol (Sudan Red G), 2,5-bis(5-tert-butyl-benzoxazol-2-yl)thiophene, 4-methoxyphenol, butylated hydroxytoluene, 2-hydroxyphenyl-s-triazine, 2-(2H-benzotriazol-2-yl)phenol, and any combination thereof.

[0462] As illustrated in the Examples section below, the use of the conjugates described herein allows for the use of such dye materials without the risk of collagen precipitation.

[0463] According to some embodiments of the present invention, the dye substance has a plurality of negatively charged groups (functional groups that are ionizable at physiological pH or at the pH of the bio-ink composition containing the dye substance). Exemplary negatively charged groups include, but are not limited to, hydroxy, sulfate, sulfonate, thiol, phosphate, phosphonate, etc.

[0464] Exemplary dye substances having multiple negatively charged groups are the polysulfate dyes.

[0465] As further demonstrated in the Examples section below, the inventors have found that vitamin B12 can be successfully utilized as a pigment material in the bio-ink compositions described herein.

[0466] According to some of the embodiments described herein, the pigment substance is vitamin B12.

[0467] According to some of the embodiments described herein, the dye substance is a quinoline.

[0468] According to some of the embodiments described herein, the dye substance is minocycline.

[0469] According to some of the embodiments described herein, the amount of the pigment material is in the range of 0.01-5% by weight, or 0.01-2% by weight, or 0.01-1% by weight, or 0.01-1% by weight, or 1-5% by weight, or 0.1-5% by weight, or 0.1-2% by weight, or 0.01-2% by weight, or 0.1-1% by weight, or 1-3% by weight (including any intermediate values ​​and subranges therebetween) of the total weight of the formulation or composition including the pigment material.

[0470] According to some of the embodiments described herein, the agent is minocycline in an amount ranging from 0.01 to 5%, or 0.01 to 2%, or 0.01 to 1%, or 0.05 to 1.5% by weight of the total weight of the minocycline-containing formulation or composition, including any intermediate or subrange therebetween.

[0471] According to one aspect of some embodiments of the present invention there is provided a curable formulation for additive manufacturing of three-dimensional objects comprising a photocurable biological material and a pigment substance capable of absorbing light at wavelengths between 300 nm and 800 nm, According to an embodiment of this aspect of the present invention, the pigment substance is or comprises vitamin B12.

[0472] According to some of the embodiments described herein of this aspect of the invention, the amount of vitamin B12 is in the range of 0.01-5% by weight, or 1-5% by weight, or 0.1-5% by weight, or 0.1-2% by weight, or 0.01-2% by weight, or 0.1-1% by weight, or 1-3% by weight (including any intermediate values ​​and subranges therebetween) of the total weight of the formulation or composition comprising vitamin B12.

[0473] The photocurable biological material can be any biological material described herein having a plurality of photocurable groups, such as (meth)acrylic groups, as described herein. Examples include, but are not limited to, (meth)acrylated gelatin, (meth)acrylated hyaluronic acid, (meth)acrylated hyaluronic acid, and (meth)acrylated collagen, such as the curable collagen described herein in any of the embodiments, and / or the conjugate described herein in any of the embodiments.

[0474] According to one aspect of some embodiments of the present invention, there is provided a process for additive manufacturing of a three-dimensional object characterized at least in part by a biological material as described herein in any of the embodiments. Additive manufacturing is a bioprinting process as described herein with respect to a bio-ink composition, comprising dispensing at least one modeling material formulation to successively form a plurality of layers in a configuration pattern corresponding to the shape of the object.

[0475] According to an embodiment of this aspect of the invention, for at least a portion of the plurality of layers, the dispensing is a dispensing of a modeling material formulation comprising a bio-ink composition comprising vitamin B12 as a pigment material, as described in any of the embodiments herein.

[0476] According to some embodiments, the additive manufacturing is DLP.

[0477] According to some embodiments, the process further comprises exposing a portion of the plurality of layers to radiation suitable for solidifying the bio-ink composition. According to exemplary embodiments, the radiation is at a wavelength described herein.

[0478] It is expected that many related curable biocompatible materials, curable biological materials, bioprinting media, and / or bioprinting techniques will be developed during the life of the patent maturing 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.

[0479] As used herein, the term "about" means ±10% or ±5%.

[0480] The terms "comprises," "comprising," "includes," "including," "having" and conjugations thereof mean "including, but not limited to."

[0481] The term "consisting of" means "including and limited to."

[0482] 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.

[0483] 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.

[0484] 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.

[0485] 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.

[0486] 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.

[0487] 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.

[0488] Throughout this specification, references to "centipoise" or "Cp" include the corresponding Pa·sec value (1 Pa·sec = 1,000 centipoise).

[0489] Throughout this specification, when the phrase "weight percent" or "wt %" or "wt%" is provided in connection with embodiments of a formulation (e.g., a modeling formulation, a curable formulation, a bio-ink composition), this phrase refers to the weight percent of the total weight of the respective uncured formulation.

[0490] Throughout this specification, acrylic materials are used generically to refer to materials having one or more acrylic acid groups, methacrylic acid groups, acrylamide groups, and / or methacrylamide groups.

[0491] Similarly, acrylic groups are used to collectively refer to hardenable groups that are acrylic acid groups, methacrylic acid groups, acrylamide groups, and / or methacrylamide groups, preferably acrylic acid groups or methacrylic acid groups (also referred to herein as (meth)acrylic acid groups).

[0492] Throughout this specification, the term "(meth)acrylic" encompasses acrylic and methacrylic materials.

[0493] Throughout this specification, the phrase "linking moiety" or "linking group" refers to a group that connects two or more moieties or groups in a compound. Linking moieties are usually derived from difunctional or trifunctional compounds and can be considered as diradical or triradical moieties connected by two or three of their atoms, respectively, to two or three other moieties.

[0494] Exemplary linking moieties include a hydrocarbon moiety or chain, as defined herein, optionally interrupted by one or more heteroatoms, and / or any of the chemical groups described below (when defined as linking groups).

[0495] When a chemical group is referred to herein as an "terminal group", it is to be understood as a substituent that is connected by one atom of it to another group.

[0496] Throughout this specification, the term "hydrocarbon" refers generally to a chemical group composed primarily of carbon and hydrogen atoms. The hydrocarbon may be composed of alkyl, alkene, alkyne, aryl, and / or cycloalkyl, each of which may be substituted or unsubstituted and may be interrupted by one or more heteroatoms. The number of carbon atoms may range from 2 to 30, and is preferably less, for example, 1 to 10, or 1 to 6, or 1 to 4. The hydrocarbon may be a linking group or a terminal group.

[0497] As used herein, the term "amine" refers to both the -NR'R'' and -NR'- groups, where R' and R'' are each independently hydrogen, alkyl, cycloalkyl, or aryl, as these terms are defined below.

[0498] Thus, the amine group can be a primary amine where R' and R'' are both hydrogen, a secondary amine where R' is hydrogen and R'' is alkyl, cycloalkyl, or aryl, or a tertiary amine where R' and R'' are each independently alkyl, cycloalkyl, or aryl.

[0499] Alternatively, R' and R'' can each independently be hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, carbonyl, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine, and hydrazine.

[0500] The term "amine" is used herein to represent the -NR'R'' group when the amine is a terminal group, as defined below, and is used herein to represent the -NR'- group when the amine is a linking group or part of a linking moiety.

[0501] The term "alkyl" refers to saturated aliphatic hydrocarbons, including straight-chain and branched-chain groups. Preferably, the alkyl group has 1-30, or 1-20 carbon atoms. When a numerical range is mentioned herein, such as "1-20," it means that the group (in this case the alkyl group) may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms. The alkyl group may be substituted or unsubstituted. A substituted alkyl can have one or more substituents, where each substituent can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine, and hydrazine.

[0502] An alkyl group can be a terminal group (as that term is defined above and is attached to a single adjacent atom) or a linking group (as that term is defined above and connects two or more moieties through at least two carbons in the chain). When an alkyl is a linking group, it is also referred to herein as an "alkylene" or an "alkylene chain."

[0503] As used herein, alkenes and alkynes are alkyl, as defined herein, containing one or more double or triple bonds, respectively.

[0504] The term "cycloalkyl" refers to an all-carbon monocyclic or fused ring (i.e., rings which share adjacent pairs of carbon atoms) group in which one or more of the rings does not have a completely conjugated pi-electron system. Examples include, but are not limited to, cyclohexane, adamantine, norbornyl, isobornyl, and the like. Cycloalkyl groups can be substituted or unsubstituted. Substituted cycloalkyls can have one or more substituents, where each substituent can be independently, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine, and hydrazine. Cycloalkyl groups can be terminal groups (as this term is defined above and is attached to a single adjacent atom) or linking groups (as this term is defined above and is connected to two or more moieties at two or more positions).

[0505] The term "heteroalicyclic" refers to a monocyclic or fused ring group having one or more atoms in the ring, such as nitrogen, oxygen, and sulfur. The ring may also have one or more double bonds. However, the ring does not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino, oxalidine, etc.

[0506] Heteroalicyclics can be substituted or unsubstituted. Substituted heteroalicyclics can have one or more substituents, where each substituent can be independently, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine, and hydrazine. A heteroalicyclic group can be a terminal group (as that term is defined above and is attached to a single adjacent atom) or a linking group (as that term is defined above and connects two or more moieties at two or more positions thereof).

[0507] The term "aryl" refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a completely conjugated pi-electron system. The aryl group may be substituted or unsubstituted. A substituted aryl may have one or more substituents, where each substituent may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine, and hydrazine. An aryl group can be a terminal group (as that term is defined above and is attached to a single adjacent atom) or a linking group (as that term is defined above and connects two or more moieties at two or more positions thereof).

[0508] The term "heteroaryl" refers to a monocyclic or fused ring (i.e., rings sharing adjacent pairs of atoms) group having one or more atoms (such as nitrogen, oxygen, and sulfur) in the ring and further having a completely conjugated pi-electron system. Examples of heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. Heteroaryl groups can be substituted or unsubstituted. Substituted heteroaryls can have one or more substituents, where each substituent can be independently, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine, and hydrazine. Heteroaryl groups can be terminal groups (as this term is defined above, attached to a single adjacent atom) or linking groups (as this term is defined above, connecting two or more moieties at two or more positions). Representative examples are pyridine, pyrrole, oxazole, indole, purine and the like.

[0509] The terms "halide" and "halo" refer to fluorine, chlorine, bromine, or iodine.

[0510] The term "haloalkyl" refers to an alkyl group, as defined above, which is further substituted with one or more halide groups.

[0511] The term "carbonyl" or "carbonate" as used herein refers to a -C(=O)-R' terminal group or a -C(=O)- linking group, as these terms are defined above, where R' is as defined herein.

[0512] The term "thiocarbonyl," as used herein, refers to a -C(=S)-R' terminal group or a -C(=S)- linking group, as these terms are defined above, where R' is as defined herein.

[0513] The term "oxo," as used herein, represents the (=O) group in which an oxygen atom is connected to an atom (eg, a carbon atom) by a double bond at the specified position.

[0514] The term "thioxo," as used herein, represents the (=S) group in which a sulfur atom is connected to an atom (eg, a carbon atom) by a double bond at the specified position.

[0515] The term "oxime" refers to either the =N-OH terminal group or the =NO- linking group, as these terms are defined above.

[0516] The term "hydroxyl" refers to an --OH group.

[0517] The term "alkoxy" refers to both -O-alkyl and -O-cycloalkyl groups as defined herein. The term alkoxide refers to a -R'O - group, where R' is as defined herein.

[0518] The term "aryloxy" refers to both an --O-aryl and an --O-heteroaryl group, as defined herein.

[0519] The term "thiohydroxy" or "thiol" refers to a -SH group. The term "thiolate" refers to a -S - Represents a group.

[0520] The term "thioalkoxy" refers to both an --S-alkyl group, and an --S-cycloalkyl group, as defined herein.

[0521] The term "thioaryloxy" refers to both an --S-aryl, and an --S-heteroaryl group, as defined herein.

[0522] "Hydroxyalkyl," also referred to herein as "alcohol," refers to an alkyl, as defined herein, substituted with a hydroxy group.

[0523] The term "acyl halide" refers to the group -(C=O)R'''', where R'''' is a halide as defined above.

[0524] As used herein, the term "carboxylate" includes C-carboxylates and O-carboxylates.

[0525] The term "C-carboxylate" refers to a -C(=O)-OR' terminal group or a -C(=O)-O- linking group, as these terms are defined above, where R' is as defined herein.

[0526] The term "O-carboxylate" refers to an -OC(=O)R' terminal group or an -OC(=O)- linking group, as these terms are defined above, where R' is as defined herein.

[0527] Carboxylates can be linear or cyclic. When cyclic, R' and the carbon atom are linked together in the C-carboxylate to form a ring, which is also called a lactone. Alternatively, R' and O are linked together in the O-carboxylate to form a ring. Cyclic carboxylates can function as linking groups, for example, when an atom in the ring formed is linked to another group.

[0528] As used herein, the term "thiocarboxylate" includes C-thiocarboxylates and O-thiocarboxylates.

[0529] The term "C-thiocarboxylate" refers to a -C(=S)-OR' terminal group or a -C(=S)-O- linking group, as these terms are defined above, where R' is as defined herein.

[0530] The term "O-thiocarboxylate" refers to an -OC(=S)R' terminal group or an -OC(=S)- linking group, as these terms are defined above, where R' is as defined herein.

[0531] Thiocarboxylates can be linear or cyclic. When cyclic, R' and the carbon atom are linked together in the C-thiocarboxylate to form a ring, and the group is also called a thiolactone. Alternatively, R' and O are linked together in the O-thiocarboxylate to form a ring. Cyclic thiocarboxylates can function as linking groups, for example, when an atom in the ring formed is linked to another group.

[0532] As used herein, the term "carbamate" includes N-carbamates and O-carbamates.

[0533] The term "N-carbamate" refers to an R"OC(=O)-NR'- terminal group or an -OC(=O)-NR'- linking group, as these terms are defined above, where R' and R'' are defined herein.

[0534] The term "O-carbamate" refers to an -OC(=O)-NR'R'' end group or an -OC(=O)-NR'- linking group, as these terms are defined above, where R' and R'' are defined herein.

[0535] Carbamates can be linear or cyclic. When cyclic, R' and a carbon atom are linked together to form a ring in an O-carbamate. Alternatively, R' and O are linked together to form a ring in an N-carbamate. Cyclic carbamates can function as linking groups, for example, when an atom in the ring formed is linked to another group.

[0536] As used herein, the term "carbamate" includes N-carbamates and O-carbamates.

[0537] As used herein, the term "thiocarbamate" includes N-thiocarbamates and O-thiocarbamates.

[0538] The term "O-thiocarbamate" refers to an -OC(=S)-NR'R'' terminal group or an -OC(=S)-NR'- linking group, as these terms are defined above, where R' and R'' are defined herein.

[0539] The term "N-thiocarbamate" refers to an R"OC(=S)NR'- terminal group or an -OC(=S)NR'- linking group, as these terms are defined above, where R' and R'' are defined herein.

[0540] The thiocarbamates can be linear or cyclic as described herein for the carbamates.

[0541] As used herein, the term "dithiocarbamate" includes S-dithiocarbamates and N-dithiocarbamates.

[0542] The term "S-dithiocarbamate" refers to an -SC(=S)-NR'R'' terminal group or an -SC(=S)NR'- linking group, as these terms are defined above, where R' and R'' are defined herein.

[0543] The term "N-dithiocarbamate" refers to an R"SC(=S)NR'- terminal group or an -SC(=S)NR'- linking group, as these terms are defined above, where R' and R" are defined herein.

[0544] The term "urea", also referred to herein as "ureido", refers to the -NR'C(=O)-NR''R''' terminal group or the -NR'C(=O)-NR''- linking group, as these terms are defined above, where R' and R'' are as defined herein and R''' is as defined herein for R' and R''.

[0545] The term "thiourea," also referred to herein as "thioureido," refers to the -NR'-C(=S)-NR''R''' terminal group or the -NR'-C(=S)-NR''- linking group, where R', R'' and R''' are as defined herein.

[0546] As used herein, the term "amide" includes C-amides and N-amides.

[0547] The term "C-amido" refers to a -C(=O)-NR'R'' terminal group or a -C(=O)-NR'- linking group, as these terms are defined above, where R' and R'' are defined herein.

[0548] The term "N-amido" refers to an R'C(=O)-NR''- terminal group or an R'C(=O)-N- linking group, as these terms are defined above, where R' and R'' are defined herein.

[0549] The amide can be linear or cyclic. When cyclic, R' and the carbon atom are linked together in the C-amide to form a ring, and the group is also called a lactam. Cyclic amides can function as linking groups, for example, when an atom in the ring formed is linked to another group.

[0550] As used herein, the term "alkylene glycol" refers to a -O-[(CR'R'') z -O] y -R''' end group or -O-[(CR'R'') z -O] y -represents a linking group, where R', R'', and R''' are as defined herein, z is an integer from 1 to 10, preferably 2 to 6, more preferably 2 or 3, and y is an integer greater than or equal to 1. Preferably, R' and R'' are both hydrogen. When z is 2 and y is 1, the group is ethylene glycol. When z is 3 and y is 1, the group is propylene glycol. When y is 2 to 4, the alkylene glycol is referred to herein as an oligo(alkylene glycol). When y is greater than 4, it is a poly(alkylene glycol). A capped poly(alkylene glycol) has R''', where R''' is other than hydrogen and can be, for example, alkyl (e.g., lower alkyl), carbonyl, and the like moieties.

[0551] 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.

[0552] 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.

[0553] Working Example 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. EXAMPLES

[0554] Synthesis of PEG with reactive and curable groups Synthesis Step I: Preparation of succinimide-capped poly(ethylene glycol) (PEG) (Compound 1): [ka]

[0555] PEG 6000 (TCI America) (10 grams, 1.66 mmol) was suspended in 40 mL of dry 1,4-dioxane, and the suspension was heated to 50° C. and stirred until completely dissolved. The resulting solution was allowed to cool to room temperature, and then N,N′-disuccinimidyl carbonate (DSC) powder (2.56 grams, 10 mmol) was added under a stream of nitrogen.

[0556] 4-(Dimethylamino)pyridine (4-DMAP, 1.23 grams, 10 mmol) was dissolved in acetone (30 mL) and the solution was added to the suspension in one portion. The suspension was stirred at room temperature under nitrogen overnight.

[0557] The resulting clear solution was evaporated to about 30 mL and poured into 150 mL of cold diethyl ether. The precipitated solid was filtered on a Buchner and suspended in ethyl acetate for 10 minutes. The resulting white solid was then filtered on a Buchner using a Whatmann filter paper No. 4 to give compound 1 as a white solid (9.34 grams). The product was dried under high vacuum and stored under argon at -20°C.

[0558] 1 H NMR (CDCl3, 500mHz, δppm): 4.41 (dd, 4H), 3.58 (s), 2.81 (s, 8H).

[0559] Preparation of methacrylated PEG (Compound 2): [ka]

[0560] Compound 1 (9.34 grams, 1.48 mmol) was suspended in 75 mL of 1,4-dioxane. The suspension was heated to 50° C. and stirred until completely dissolved. The solution was allowed to cool to room temperature. 3-Aminopropylmethacrylamide hydrochloride (265.3 mg, 1.48 mmol) was dissolved in 10 mL of mQ water, to which 4-DMAP (366 mg, 2.97 mmol) was added and the resulting aqueous solution was stirred until completely dissolved. The pH of the solution was 10.15. The aqueous solution was added dropwise to the solution of compound 1. The solution became cloudy after 4 mL was added. 3 mL of mQ water was added to aid in dissolution. The addition was continued over 30 minutes and the solution was further stirred at room temperature overnight. TLC (using ninhydrin reagent as developer) showed no free amine was present. Toluene (200 mL) was added and the flask was fitted with a Dean-Stark apparatus. The reaction was refluxed until no more water was distilling off.

[0561] The Dean-Stark apparatus was removed and the volume of toluene was reduced to approximately 30 mL in a rotavaporizer. The solution was then poured into 180 mL of cold diethyl ether, the resulting precipitate was filtered on a Büchner, and the white solid was redissolved in 30 mL of dichloromethane (DCM) and precipitated by adding 180 mL of cold diethyl ether. The resulting white solid was filtered on a Büchner using a Whatmann filter paper No. 4 to give compound 2 as a white solid (7.32 grams). The product was dried under high vacuum and stored under argon at -20°C.

[0562] 1 H NMR(CDCl3,500mHz,δppm):6.62(m,1H) 5.74(s,1H) 5.33(s+m,2H) 4.21(m,2H) 3.37(dd,2H) 3.24(dd,2H) 1.98(s,3H) 1.69(q,2H).

[0563] Preparation of nitrobenzoyl-capped methacrylated PEG (compound 3): [ka]

[0564] Compound 2 (7.29 grams, 1.17 mmol) was dissolved in 25 mL of dry DCM.

[0565] 4-Nitrobenzoyl chloride (237 mg, 1.17 mmol) was dissolved in 2 mL of dry dichloromethane (DCM) and the solution was added in one portion to the solution containing compound 2.

[0566] 4-DMAP (145 mg, 1.17 mmol) was dissolved in 2 mL of dry DCM and the solution was added to the reaction mixture. The resulting solution was stirred overnight at room temperature under nitrogen atmosphere and then poured into 180 mL of diethyl ether. The resulting white precipitate was filtered on a Buchner using Whatmann filter paper No. 1, then recrystallized in 150 mL of ethyl acetate, filtered on a Buchner, and suspended in ethyl acetate for 10 minutes. The white solid was filtered on a Buchner using Whatmann filter paper No. 4 to give compound 3 as a white solid (6.63 grams). The product was dried under vacuum and stored at -20°C under argon.

[0567] 1 H NMR(CDCl3,500mHz,δppm):8.29(d,2H) 7.38(d,2H) 6.62(m,1H) 5.74(s,1H) 5.33(s+m,2H) 4.45(m,4H) 4.21(m,4H) 3.36(dd,2H) 3.24(dd,2H) 1.98(s,3H) 1.66(q,2H). *The 2.89 singlet represents residual unreacted NHS carbonate.

[0568] Synthesis Step II: Preparation of nitrobenzoyl protected PEG (compound 11): [ka]

[0569] PEG 6000 (20 grams, 3.33 mmol) was dried in a vacuum oven at 120° C. and 0.1 mbar overnight, then allowed to cool to room temperature and dissolved in 50 mL of dry DCM.

[0570] 4-Nitrophenyl chloroformate (1.34 grams, 6.66 mmol) was dissolved in 5 mL of dry DCM, the solution was added to the PEG solution in one portion, and the resulting reaction mixture was stirred for 5 minutes.

[0571] 4-DMAP (821 mg, 6.66 mmol) was dissolved in 5 mL of DCM and the solution was added dropwise to the reaction mixture, which was then stirred at room temperature overnight.

[0572] The solvent volume was reduced to about 30 mL by evaporation, and the remaining solution was poured into 200 mL of diethyl ether. The precipitated solid was collected by filtration on a Buchner funnel, redissolved in 40 mL of DCM, and poured into 200 mL of diethyl ether. The resulting solid was collected by Buchner filtration and dried under vacuum to give compound 1 (19.35 grams) as a white solid.

[0573] 1 H NMR (CDCl3, 500mHz, δppm): 8.20 (d, 2H), 7.32 (d, 2H), 4.37 (m, 2H), 3.57 (s, 545H).

[0574] Preparation of methacrylated PEG (Compound 12): [ka]

[0575] Compound 11 (19.35 grams, 3.03 mmol) was dissolved in 180 mL of acetonitrile. 3-Aminopropyl methacrylate HCl (544 mg, 3.05 mmol) was dissolved in 25 mL of mQ water, then 4-DMAP (752 mg, 6.10 mmol) was added and the solution was stirred until completely dissolved. The resulting solution was then added dropwise to the solution of compound 11 over 1 hour, and the resulting reaction mixture was stirred at room temperature. The solvent volume was then reduced to approximately 80 mL by evaporation, and 150 mL of DCM was added. MgSO4 was added to dry the solution, filtered, and the solvent volume was reduced to approximately 30 mL. The solution was poured into 180 mL of diethyl ether and the precipitate was collected by Buchner filtration. The white solid was redissolved in 50 mL of DCM, the solution was poured into 200 mL of diethyl ether, and the resulting white solid was collected by filtration to give compound 12 as a white solid (16.766 grams).

[0576] 1 H NMR (CDCl3,500mHz,δppm):5.70(s,1H),5.27(s,1H),4.15(m,2H),3.57(s,545H),3.30(dd,2H),3.17(dd,2H),1.91(s,3H),1.62(q,2H).

[0577] Preparation of nitro-benzoyl capped methacrylated PEG (compound 3): [ka]

[0578] Compound 12 (16.76 grams, 2.71 mmol) was dissolved in 50 mL of dry DCM. 4-Nitrophenyl chloroformate (655 mg, 3.25 mmol) was dissolved in 5 mL of dry DCM and the solution was added to the reaction mixture in one portion followed by stirring for 5 minutes.

[0579] 4-DMAP (334 mg, 2.71 mmol) was dissolved in 5 mL of DCM and the solution was added dropwise to the reaction mixture which was then stirred at room temperature overnight.

[0580] The solvent volume was reduced to about 30 mL by evaporation, and the solution was poured into 200 mL of diethyl ether. The precipitated solid was collected by filtration on a Buchner funnel, redissolved in 40 mL of DCM, and poured into 200 mL of diethyl ether. The resulting solid was collected by Buchner filtration and dried under vacuum to give compound 3 as a white solid (about 14 grams).

[0581] 1 H NMR(CDCl3,500mHz,δppm):8.21(d,1H),7.33(d,1H),5.70(s,1H),5.27(s,1H),4.38(dd,1H) 4.15(m,2H),3.57(s,545H),3.30(dd,2H),3.17(dd,2H),1.91(s,3H),1.62(q,2H).

[0582] Synthetic Step III: [ka]

[0583] PEG 6000 (20 grams, 3.33 mmol) was placed in a 500 mL amber round bottom flask and 240 mL of toluene was added. The flask was fitted with a Dean-Stark apparatus and residual water was allowed to distill off for 4 hours.

[0584] The solution was allowed to cool to room temperature. 4-Nitrophenyl chloroformate (2.68 grams, 13.32 mmol) was added directly along with triethylamine (1.85 mL, 13.32 mmol) and the reaction mixture was heated to 60° C. overnight. The solvent volume was reduced to approximately 30 mL by evaporation and the remaining solution was poured into 200 mL of diethyl ether. The precipitated solid was collected by filtration on a Buchner funnel, redissolved in 40 mL of DCM and poured into 200 mL of diethyl ether. The resulting solid was collected by Buchner filtration and dried under vacuum to give compound 11 (19.35 grams) as a white solid.

[0585] 1H NMR (CDCl3, 500mHz, δppm): 8.20 (d, 4H), 7.32 (d, 4H), 4.37 (m, 4H), 3.57 (s, 545H).

[0586] [ka]

[0587] Compound 11 (19.35 grams, 3.03 mmol) was dissolved in 160 mL of acetonitrile in a 250 mL round bottom flask made of amber glass. 3-Aminopropyl methacrylate HCl (544 mg, 3.05 mmol) was dissolved in 5 mL of mQ water, then triethylamine (850 μL, 6.10 mmol) was added and the solution was stirred until completely dissolved. The resulting aqueous solution was then added dropwise to the solution of compound 11 over a period of 2 hours, and the resulting reaction mixture was stirred overnight at room temperature. The solvent volume was then reduced to approximately 30 mL by evaporation, and 40 mL of toluene was added. The flask was fitted with a Dean-Stark apparatus and 5 mL of water was distilled off. The solution was then evaporated to reduce the solvent volume to approximately 30 mL and poured into 200 mL of cold diethyl ether. The precipitate was collected by Buchner filtration. It was then redissolved in 40 mL of toluene and poured into 300 mL of cold diethyl ether. Compound 3 was collected by Buchner filtration and dried under vacuum overnight to give 15.75 grams as a white solid.

[0588] 1 H NMR(CDCl3,500mHz,δppm):8.24(d,2H),7.36(d,2H),5.70(s,1H),5.31(s,1H),4.38(dd ,2H)4.15(m,2H),3.57(s,545H),3.30(dd,2H),3.17(dd,2H),1.95(s,3H),1.65(q,2H). EXAMPLES

[0589] Collagen-methacrylated PEG conjugate Synthesis: 727 mL of recombinant human type I collagen (Collplant Ltd.) (2.9 mg / mL) was placed in a 1 liter single jacketed reactor pre-cooled to 4° C., to which 80 mL of 2 M MOPS buffer (pH 8.0) was added along with 34 mL of 4 M NaCl. The solution was adjusted to pH 8.0 using 10 N NaOH solution and then cooled to 6° C. 9.3 grams of compound 3 (1:2 molar ratio to collagen lysine residues) was dissolved in 10 mL of mQ water. The resulting solution was added in one portion to the cooled (6° C.) collagen solution with stirring, and the reaction mixture was stirred at 6° C. for 18 hours. The reaction was then quenched by adding 150 mL of 1 M HCl, resulting in a solution with pH 3. The product was purified by dialysis. The dialysis was performed 11 times with 800 mL of 10 mM HCl by repeatedly adding 800 mL of 10 mM HCl and reducing the volume in the vessel to 800 mL. The solution was then concentrated to a void volume of 65 mL.

[0590] Figure 1 shows a schematic of the reaction product of compound 3 (PEG-MA) with collagen. The PEG-methacrylate moiety is attached to a lysine residue on collagen via a carbamate bond.

[0591] FIG. 2 shows SDS-PAGE comparing collagen and CPM.

[0592] SDS-PAGE analysis suggests that at least 2% of the lysine residues, e.g., 2-10% of the lysine residues, have PEG-MA moieties attached.

[0593] Characterization: Three formulations were prepared, each containing 7% PEG-DA 3400 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photoinitiator. One formulation further contained methacrylated recombinant human type I collagen (CMR) at a concentration of 4 mg / mL, one formulation further contained the reaction product of compound 3 (PEG-MA) with collagen (also referred to as "CPM") shown in Figure 1 at a concentration of 4 mg / mL, and one served as a protein-free control.

[0594] Viscosity measurements were performed using a UHP steel cone-plate (40 mm 1°) geometry, with samples conditioned at 22°C, with a 10 s soak time and 20 s preshear at 2.0 rad / s. This was followed by a Flow Sweep (logarithmic) test applied at shear rates of 0.01-1000 (1 / s), 3 points per decade. A maximum equilibration time of 60 s was used to detect steady state, with a sampling period of 5 s (5% tolerance). The applied force was removed 1, 3 and 5 min after the initial measurement, then the viscosity was remeasured. Sample volume: 300 microliters, containing 9.3 mg / mL of CPM or CMR in 10 mM HCl buffer solution (pH 2.5).

[0595] The data obtained are shown in FIG. 3A (for the CPM formulation) and FIG. 3B (for the CMR formulation).

[0596] As can be seen, the CPM sample retains its viscosity even after 1 minute of relaxation, while the viscosity of the CMR drops significantly and does not recover even after 5 minutes of relaxation.

[0597] Recovery under shear (shear recovery) was tested as follows: Flow peak measurements were performed using a Discovery HR 2 (TA Instruments) with UHP steel cone plate (40mm 1°) geometry, with samples conditioned at 22°C, soak time 10 seconds, and pre-sheared at 2.0 rad / sec for 20 seconds. Flow peak retention (log) tests were then performed at a shear rate of 1.0 (1 / sec) for 200 seconds, followed by a shear rate of 100 (1 / sec) for 100 seconds, followed by a shear rate of 1.0 (1 / sec) for 100 seconds. Sample volume: 300 microliters.

[0598] Figures 4A-B show data showing the change in viscosity as shear force is manipulated for the formulations containing CPM (Figure 4A) and CMR (Figure 4B) described above, showing that CPM is more viscous than CMR, but also has higher recovery under shear (97% for CPM vs. 87% for CMR), indicating that PEGylated collagen is significantly more resistant to shear force than CMR.

[0599] The photorheological characterization of all the scaffolds obtained upon curing of the aforementioned formulations was tested using a Discovery HR-2 rheometer equipped with a 20 mm parallel plate geometry and an Omnicure (series 2000) optical attachment as light source. 40 μL of each sample was loaded onto the lower plate and the upper geometry was lowered to give a gap size of 100 μm. The measurement duration was set to 120 s at an angular frequency of 2 Hz and a strain of 1%, in which the samples were preconditioned for 30 s and then irradiated with 22 mW / cm. 2 The collagen was then exposed to light for 6 seconds at 400 nm and the light was initiated at 400 nm for 6 seconds. The data obtained are shown in Figure 5. As can be seen, the storage modulus, and correspondingly the elastic modulus, of CPM was lower than that of CMR, indicating that the introduction of PEG moieties into the prepolymerized collagen improved the elasticity.

[0600] Solubility: DLP bioprinting generally requires the use of ink formulations containing dyes that absorb well in the 365-405 nm range, a requirement for printing microstructures with 10 micrometer resolution. Most water-soluble dyes available for DLP contain sulfate or other negatively charged groups that confer water solubility.

[0601] Since collagen is a positively charged protein with multiple positively charged groups, the presence of sulfates, or dyes with other negatively charged groups, can lead to physical cross-linking or simple precipitation (following the Hofmeister series). This creates severe limitations and forces the use of dyes that are water-soluble, biocompatible, and non-toxic, have desirable absorption properties, and do not have negatively charged groups. Such dyes are not readily available, and when used, large quantities are required to obtain good resolution without precipitation.

[0602] Thus, as can be seen in the left vial of Figure 6, when a 10 mg / mL solution of CMR is mixed with 1 gram of UV386a, an exemplary polysulfate dye, precipitation is observed.

[0603] As can be further seen in the right vial of Figure 6, when a 10 mg / mL solution of CPM was mixed with 1 gram of UV386a, the solution remained clear and there was no significant precipitation. Without being bound to any particular theory, this phenomenon can be explained by the high polarity of the PEG moiety, which enhances the solubility of the protein even in the presence of polysulfate, and / or by the interference of the PEG moiety with the physical cross-linking between the sulfate groups and the positively charged groups of collagen.

[0604] The optical density of the two solutions tested was measured at 600 nm: the OD of the CMR solution was 2.181, and the OD of the CPM solution was 0.294, almost an order of magnitude apart.

[0605] This allows the use of negatively charged dyes, circumventing the limitations imposed in selecting suitable dyes.

[0606] Preparation of methacrylated collagen-methacrylated PEG conjugate: The present inventors have also devised a "hybrid" conjugate in which the collagen-methacrylated PEG conjugate described above was further modified by covalently attaching methacrylic acid groups directly to the collagen as follows.

[0607] 10 mL of CPM (3.0 mg / mL) prepared as described above was added to a 20 mL amber flask. Then, 1.4 mL of 2 M MOPS buffer solution (pH=7.5) was added, followed by 400 microliters of 4 M NaCl. The solution was cooled to 4° C., and then 1.5 microliters of methacrylic anhydride was added. The solution was stirred overnight at 4° C. The degree of modification was shown to be 42% by TNBS assay. EXAMPLES

[0608] The present inventors have devised the use of Vitamin B12 as a light absorbing dye in the DLP process.

[0609] Vitamin B12 (180 mg) or 4-nitrophenol (43 mg) was dissolved in 6 mL of mQ water along with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (200 mg). One gram of PEG(6000)-diacrylate was added to the solution, followed by the CMR described herein to obtain a final concentration of 5 mg / mL of CMR, and 10 mM HCl was added to make the total volume 20 mL. The resulting mixtures were each stirred until a homogenous solution was obtained and used to 3D print (using a DLP printer) hydrogels with 50 μm channels along the Z axis and 150 μm pores in the XY plane. DLP printing with both formulations was performed without issue.

[0610] The viscosities of the two formulations were evaluated and compared. Measurements were performed on a Discovery HR-2 rheometer equipped with a 40 mm cone plate at 1°. 300 μL of sample was loaded into the lower geometry and the upper geometry was lowered to give a gap of 32 μm. The temperature was maintained at 22°C and viscosity values ​​were taken from shear rates ranging from 0.01 to 1000 (1 / s) (3 points per decade). The data obtained are shown in Figure 7.

[0611] As can be seen, at shear rates up to at least 10 (1 / sec), the Vitamin B12 formulation exhibits a much lower viscosity compared to the 4-nitrophenol formulation, making it more suitable for bioprinting applications. This is in addition to the inherent advantage of using naturally derived Vitamin B12 as a pigment that is easily washed off the printed object without having to meet hazardous materials regulatory requirements. EXAMPLES

[0612] To test the effect of the amount of photoinitiator (PI), LAP, three different formulations were prepared that differed only in LAP concentration.

[0613] A formulation containing 0.5% LAP was prepared as follows.

[0614] 900 mg of LAP was dissolved in 9 mL of Milli-Q water, to which was added 1 gram of PCL(2000)-PEG(20K)-PCL-(2000) diacrylate, 0.1 gram of PEG-DA 3400, and 900 mg of PEG-DA 700. 3.0 mL of 10 mM HCl was then added, and the solution was stirred until a clear solution was obtained. 5.8 grams of CMR (16.9 mg / mL) solution was then added, resulting in a 5 mg / mL solution.

[0615] Formulations containing 0.75% and 0.9% LAP were similarly prepared using the respective amounts of LAP.

[0616] The viscosity of each formulation was measured as described above, and the resulting data is shown in Figure 8. As can be seen, the amount of PI mainly affects the viscosity of the formulation at shear rates up to 10 (1 / sec), with lower amounts of PI resulting in lower viscosities. EXAMPLES

[0617] The inventors have envisioned the use of minocycline as a light absorber (e.g., a dye material as described herein) in 3D bioprinting and tested its performance in a DLP 3D bioprinting process using a collagen-containing curable formulation.

[0618] Exemplary tests used a curable formulation including curable rh collagen having multiple methacrylic acid groups (also referred to herein as CMR), an aqueous carrier, one or more additional curable polymeric materials (e.g., PEG-DA), and a photoinitiator as described in WO 2018 / 225076.

[0619] In an exemplary procedure, minocycline (Apollo Scientific) was dissolved in mQ water along with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, photoinitiator). PEG(6000) diacrylate was added to the solution, followed by CMR and dilute HCl. The resulting mixtures were stirred until a homogenous solution was obtained, and used to 3D print (using a DLP printer) hydrogels with 200 μm channels along the Z axis and in the XY plane.

[0620] The effect of minocycline concentration on the rate and extent of setting of the formulations was examined by determining the G' value of each formulation during the hardening stage.

[0621] Measurements were performed using a Discovery HR-2 rheometer equipped with a 20 mm parallel plate geometry and an Omnicure (series 2000) optical attachment as light source. 25–90 μL of each sample was loaded onto the lower plate and the upper geometry was lowered to give a gap size of 50–250 μm depending on the drop volume. Measurement duration was set to 120 s at an angular frequency of 2 Hz and strain of 1%, in which the samples were preconditioned for 30 s and then irradiated using an external UV light source at 365 nm at 50 mW / cm. 2 The cells were then illuminated with light for 60 seconds.

[0622] Formulations containing 0, 0.04, 0.08, 0.12, 0.16, and 0.2% by weight minocycline were tested, and the results are shown in Figure 9.

[0623] As can be seen, all formulations reached maximum crosslinking levels, indicating that at the high light energy used, minocycline does not affect crosslinking of the formulations even at very low concentrations of 0.08 wt %.

[0624] No precipitation was observed after inclusion of minocycline, indicating that minocycline is suitable as a light absorber collagen-containing curable formulation as described herein. EXAMPLES

[0625] As shown in FIG. 10, a conjugate was synthesized that included collagen (rh collagen as described herein) with multiple methacryl groups attached to some of the lysine residues and multiple PEG moieties (methoxy-capped, non-curable).

[0626] Briefly, 209.3 grams of MOPS buffer was dissolved in 200 mL of mQ water. The solution was adjusted to pH 7.5 by adding 42 mL of 10 N NaOH and brought to a volume of 500 mL by adding mQ water. The solution was filtered through a 0.45 μm filter. 10 mL of 1 M HCl was added to 990 mL of mQ water and the solution was stirred for 5 minutes. 70 mL of recombinant human collagen type I (Collplant Ltd.) (20.3 mg / mL) was poured into 420 mL of 10 mM HCl and the solution was stirred and transferred to a 1 liter reactor and cooled to 6°C. 23 mL of 4 M NaCl was added, followed by 53 mL of MOPS 2 M buffer (pH 7.5) and the solution was adjusted to pH 8.0 by slowly adding 10 N NaOH (5.8 mL total). 5.14 grams of MeO-PEG 5000-PNC (2 mol equivalents relative to lysine residues, see FIG. 10) was dissolved in 50 mL of mQ water, the solution was added to the reactor, and the reaction mixture was stirred at 6° C. for about 24 hours.

[0627] Next, 111 microliters of methacrylic anhydride was added and the reaction was stirred at 6° C. for approximately 24 hours. The reaction was then quenched by adding 115 mL of 1 M HCl to give a solution with a pH of 2.5. The product was purified by dialysis, which was performed 10 times with 500 mL of 10 mM HCl by repeatedly adding 500 mL of 10 mM HCl and reducing the volume in the vessel to 500 mL.

[0628] The resulting formulation has a viscosity of about 100 centipoise, a G' of about 46,000 Pa, and a shear recovery of about 93%, all determined as described herein.

[0629] 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.

[0630] It is the intention of the applicants that all publications, patents, and patent applications mentioned herein be incorporated by reference in their entirety as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference 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. Additionally, any priority documents of this application are hereby incorporated by reference in their entirety as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference herein.

Claims

1. A conjugate comprising collagen and a plurality of elastic moieties covalently bonded to said collagen, at least some of said elastic moieties having hardenable groups.

2. The conjugate of claim 1 , wherein the curable group is at the end of each of the elastic moieties.

3. The conjugate of claim 1 , wherein the curable group is a (meth)acrylic group.

4. The conjugate described in claim 2, wherein the curable group is a (meth)acrylic group.

5. The conjugate of claim 1 , wherein at least a portion of the elastic moieties are poly(alkylene glycol) moieties.

6. 2. The conjugate of claim 1, wherein at least some or each of the elastic moieties comprises a poly(alkylene glycol) moiety terminated with an acrylic or (meth)acrylic group.

7. The conjugate of claim 1 , wherein at least a portion of the elastic moieties are covalently bonded to lysine residues of the collagen.

8. 8. The conjugate of claim 7, wherein 1-20% of the lysine residues in the collagen have the elastic moiety covalently attached thereto.

9. 8. The conjugate of claim 7, wherein at least a portion of the elastic moieties are attached to the lysine residues by carbamate bonds.

10. The conjugate of claim 1 , wherein the collagen has multiple photocurable groups.

11. 2. The conjugate of claim 1, wherein the collagen is a recombinant collagen of plant origin.

12. 2. The conjugate of claim 1, wherein the collagen is plant-derived recombinant human type I collagen.

13. The conjugate described in claim 12, wherein the collagen is a plant-derived recombinant human type I collagen having multiple photocurable groups.

14. A curable formulation comprising a conjugate according to any one of claims 1 to 13 and an aqueous carrier.

15. 15. The curable formulation of claim 14, further comprising at least one additional curable material.

16. 16. The curable formulation of claim 15, wherein the additional material is or comprises a poly(alkylene glycol) terminated with at least one (meth)acrylic group.

17. 15. The curable formulation of claim 14, wherein the curable groups included in the elastic portion are photocurable groups, and the formulation further comprises a photoinitiator.

18. 15. The curable formulation of claim 14, further comprising a pigment material capable of absorbing light at wavelengths between 300 nm and 800 nm.

19. 20. The curable formulation of claim 18, wherein the colorant material has a plurality of negatively charged groups.

20. 19. The curable formulation of claim 18, wherein said color substance is selected from the group consisting of UV386a, vitamin B12, quinoline, and minocycline.

21. 1. A process for additive manufacturing of a three-dimensional object characterized in that at least a portion thereof is a collagen-based material, the process comprising successively forming a plurality of layers in a structural pattern corresponding to a shape of the object; At least a portion of the plurality of layers are formed from a modeling material formulation comprising the curable formulation of claim 14; A process by which the three-dimensional object is produced.

22. A three-dimensional biological object using a collagen-based material in at least a portion thereof, the three-dimensional biological object comprising, in at least a portion thereof, a hardened product of the hardenable composition described in claim 14.

23. 23. The three-dimensional biological object of claim 22 for use in repairing damaged tissue and / or as an artificial tissue or organ.

24. 1. A process for additive manufacturing of a three-dimensional object characterized in that the object is at least partially composed of a collagen-based material, comprising: Choosing additive manufacturing technologies; preparing a modeling material formulation by combining collagen having at least a plurality of photocurable groups, a photoinitiator, optionally an aqueous carrier, and optionally other curable and / or non-curable components, wherein the amount of photoinitiator is selected to provide a viscosity suitable for said additive manufacturing technique; and Successively forming a plurality of layers in a structural pattern corresponding to the shape of the object. Including, at least a portion of the plurality of layers are formed from the modeling material formulation including collagen having the plurality of photocurable groups; A process by which the three-dimensional object is produced.

25. 25. The process of claim 24, wherein the collagen is human type I collagen.

26. 25. The process of claim 24, wherein the collagen is a recombinant collagen of plant origin.

27. 25. The process of claim 24, wherein the collagen is plant-derived recombinant human type I collagen.

28. The process of any one of claims 24 to 27, wherein the photoinitiator is an acylphosphine oxide type photoinitiator.

29. A curable formulation comprising: collagen having a plurality of photocurable groups covalently bonded thereto; an additional curable material having at least one photocurable group; a photoinitiator; and a pigment substance capable of absorbing light at wavelengths between 300 nm and 800 nm.

30. The curable formulation of claim 29, wherein the pigment material has a plurality of negatively charged groups.

31. The collagen is a plant-derived recombinant human collagen, and / or the additional curable material is or comprises a poly(alkylene glycol) moiety terminated with an acrylic or (meth)acrylic group; and / or the photoinitiator is an acylphosphine oxide type photoinitiator, and / or the color substance is or comprises minocycline, and / or 30. The hardenable formulation of claim 29, wherein said color substance is or comprises vitamin B12.

Images