Light-absorbing substances suitable for use in additive manufacturing and methods of identification thereof

Abstract

Photoblocker usable in additive manufacturing a three-dimensional object are disclosed. Photocurable formulations comprising the photoblockers are also disclosed. The photoblocker can be beneficially included in formulations usable in additive manufacturing of 3D objects featuring a biological material in at least a portion thereof.

Description

[0001] LIGHT-ABSORBING SUBSTANCES SUITABLE FOR USE IN ADDITIVE MANUFACTURING AND METHODS OF IDENTIFICATION THEREOF

[0002] RELATED APPLICATION

[0003] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 696,908 filed on 20 September 2024, U.S. Provisional Patent Application No. 63 / 696,902 filed on 20 September 2024. This application also incorporates by reference a PCT application having title “Formulations and designs for degradable soft tissue implants”, filed on even date with the present application and having a same applicant and having docket number 104970. The contents of all of these applications are incorporated herein by reference in their entirety. In particular, the PCT application describes collagen-based formulations usable in additive manufacturing of degradable scaffolds and / or soft tissue implants that feature mechanical properties suitable for breast implants, and to design thereof, when carrying out some embodiments of the invention.

[0004] SEQUENCE LISTING STATEMENT

[0005] The XML file, entitled 104968.xml, created on 03 September, 2025 comprising 57,344 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to additive manufacturing, and more particularly, but not exclusively, to light-absorbing substances that can be beneficially included in formulations usable in additive manufacturing of 3D objects featuring a biological material in at least a portion thereof.

[0008] Additive manufacturing (AM) is generally a process in which a three-dimensional (3D) object is manufactured utilizing a computer model of the objects. The basic operation of any AM system consists of slicing a three-dimensional computer model into thin cross sections, translating the result into two-dimensional position data and feeding the data to control equipment which manufacture a three-dimensional structure in a layerwise manner.

[0009] Various AM technologies exist, amongst which are stereolithography, digital light processing (DLP), and three-dimensional (3D) printing such as 3D inkjet printing. Such techniques are generally performed by layer-by-layer deposition and / or hardening (e.g., solidification by exposure to light,) of one or more building materials, which typically include photopolymerizable (photocurable) materials.

[0010] Stereolithography, for example, is an additive manufacturing process which employs a liquid ultraviolet (UV)-curable building material and a UV laser. In such a process, for each dispensed layer of the building material, the laser beam traces a cross-section of the part pattern on the surface of the dispensed liquid building material. Exposure to the UV laser light cures and solidifies the pattern traced on the building material and joins it to the layer below. After being built, the formed parts are immersed in a chemical bath in order to be cleaned of excess building material and are subsequently cured in a UV oven.

[0011] Digital Light Processing (DLP) printing of hydrogels is an additive manufacturing technique that uses digital light projection to create three-dimensional structures from building materials. The process involves preparation of a photosensitive building material (e.g., mixing a polymer (polymerized by free-radical polymerization) network with a photosensitive agent, such as a photoinitiator). The prepared building material is placed in a vat or reservoir, and a digital micromirror device (DMD) projects light patterns onto the building material surface. The DMD selectively illuminates specific regions of the building material, causing the photosensitive agent to initiate polymerization and solidify the liquid hydrogel layer by layer. The DLP printer builds the 3D structure by repeating the exposure and polymerization process layer by layer. As each layer solidifies, the build platform moves downward or upward (dependent on the type of printer), enabling the next layer to be added on top of the previous one. After the printing is complete, the hydrogel-based 3D structure may undergo post-processing steps such as washing to remove any unreacted material, additional curing or crosslinking, and possibly the incorporation of bioactive substances or cells for specific applications. DLP printing of hydrogels offers high resolution and the ability to create intricate and complex structures. The building material is preferably liquid when maintained in the vat and at the working temperature at which layers are formed, and are subsequently hardened / cured, typically upon exposure to light. DLP printing technology provides improved mechanical properties, scalability and robustness. Successful printability in DLP requires a carefully balanced formulation that supports accurate light-based curing, controlled flow behavior, and consistent layer adhesion throughout the printing process.

[0012] In DLP bioprinting, printability is governed by the precise interaction between the bioink’s optical, mechanical, and rheological properties and the layer-by-layer photopolymerization process. The formulation must have suitable viscosity to ensure uniform spreading of each layer, while maintaining structural stability during printing. Critically, the photoreactive components must enable rapid and consistent crosslinking upon exposure to light at defined wavelengths, ensuring high resolution and fidelity in complex geometries. Additionally, the formulation must be optimized to prevent over-curing or light scattering, which can affect pore definition and feature accuracy. In DLP bioprinting, polymerization kinetics play a central role in determining the accuracy, resolution, and mechanical integrity of the printed structure. The crosslinking reaction must proceed at a controlled rate - fast to enable rapid layer solidification and maintain printing efficiency, yet slow to prevent over-curing, which can lead to loss of detail or pore closure. Properly tuned kinetics ensure uniform curing across the entire layer and between successive layers, supporting consistent mechanical performance and dimensional stability.

[0013] In three-dimensional printing processes, such as 3D inkjet printing, a building material is dispensed from a dispensing head having a set of nozzles or nozzle arrays to deposit layers on a receiving substrate. Depending on the building material, the layers may then be cured or solidified using a suitable device.

[0014] The building materials may include modeling material formulation(s) and support material formulation(s), which form, upon hardening, the object and the temporary support constructions supporting the object as it is being built, respectively. The modeling material formulation(s) is / are deposited to produce the desired object and the support material formulation(s) is / are used, with or without modeling material elements, to provide support structures for specific areas of the object during building and assure adequate vertical placement of subsequent object layers, e.g., in cases where objects include overhanging features or shapes such as curved geometries, negative angles, voids, and so on.

[0015] Both the modeling and support material formulations typically feature a viscosity that allows maintaining in a vat and forming layers therein upon layer-wise exposure to light or dispensing / depositing, and upon being dispensed and optionally exposed to curing / hardening, feature a higher viscosity. Both the modeling and support materials are preferably liquid at the working temperature at which layers are formed or dispensed, and are subsequently hardened, typically upon exposure to hardening or curing condition such as curing energy (e.g., UV curing), to form the required layer shape. After printing is completed, support structures, if present, are removed to reveal the final shape of the fabricated 3D object. The hardening (curing) of the dispensed materials typically involves polymerization (e.g., photopolymerization) and / or crosslinking (e.g., photocrosslinking).

[0016] Additive manufacturing was first used in biological applications for forming three- dimensional sacrificial resin molds in which 3D scaffolds from biological materials were created.

[0017] 3D bioprinting is an additive manufacturing methodology which uses biological materials, optionally in combination with chemicals and / or cells, that are printed layer-by-layer with a precise positioning and a tight control of functional components placement to create a 3D structure. Three dimensional (3D) bioprinting is gaining momentum in many medicinal and aesthetic applications, especially in regenerative medicine, in order to address the need for complex scaffolds, tissues and organs suitable for transplantation.

[0018] Inherent to 3D printing in general is that the mechanical properties of the printing media (the pre-printed building material) are very different from the post-printed cured (hardened) material.

[0019] To allow tight control of the curing (e.g., polymerization) following printing, the building material commonly includes polymerizable (e.g., photopolymerizable) moieties or groups that polymerize (e.g., by chain elongation and / or cross-linking) upon curing, so as to preserve the geometric shape and provide the necessary physical properties of the final product.

[0020] Different technologies have been developed for 3D bioprinting, including 3D Inkjet printing, Extrusion printing, Laser-assisted 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 technology has its different requirements for the building material (also referred to herein as printing media), which is derived from the specific application mechanism and the curing / gelation process required to maintain the 3D structure of the scaffold post printing.

[0021] Most of the 3D bioprinting technologies employ photopolymerizable materials and electromagnetic irradiation as the curing condition to which the building material is exposed. The printing media in such technologies typically include a photoinitiator, which is capable of at least partially penetrating into voids between the hardened material, and is also capable of spreading onto the exterior surface of the hardened material. The photo-initiator is responsible for the initiation of the polymerization process when it is exposed to light with the right wavelength and energy level.

[0022] Exposing to electromagnetic irradiation is sometimes associated with selectivity issues, due to absorbance of the light energy by non-patterned building materials, which may lead to inaccurate parts, shapes and / or rough part edges. One way to overcome these limitations is the use of a photoabsorber, or a photoblocker, which is also referred to herein as a dye substance or as a light-absorbing substance, which is capable of absorbing light at a respective wavelength range, and thereby improve the resolution and achieve defined porosity and channels of scaffolds. The photoabsorber is typically responsible for reducing the light penetration mainly in the Z axis (through the printed layers) but also increases the required energy levels for the polymerization process in XY plane. WO 2023 / 073711 teaches that Vitamin B 12, UV386, minocycline and Quinoline yellow can be used as an absorbing dye substance in bioink compositions comprising recombinant collagen.

[0023] Photoabsorbers usable in additive manufacturing in general and in 3D bioprinting in particular, are described, for example, in Zhang et al., Bums Trauma. 2022; 10: tkacOlO; WO 2022 / 093236; U.S. Patent Application Publication Nos. 2020 / 339925 and 2021 / 229364; U.S. Patent No. 10,597,289; CN 114958079; WO 2023 / 073711.

[0024] Additional background art includes CN Patent Application No. 112062878; US Patent Application No. 20220010041, He, Xiangnan et al. Soft Matter (2023), 19(20), 3700-3710; Mauguiere-Guyonnet et al., Progress in Organic Coatings (2007), 59(1), 37-45; George, Beatrice et al., Double Liaison— Physique, Chimie & Economic des Peintures & Adhesifs (2002), 527, 40- 48; and Tan et al., Agricultural Sciences in China (2006), 5(11), 855-858.

[0025] Additional background art includes U.S. Patent Application Publication No. 2017 / 143831 ; WO 2018 / 225076; U.S. Patent application Publication No. 2018 / 0193524; WO 2015 / 032985; Drzewiecki et al. (2014) Langmuir, 30 31), 11204-11211; and Ravichandran et al. (2015) Journal of Materials Chemistry B, 4(2), 318-326; Gaudet & Shreiber (2012) Biointerphases, 7(1), 25. SUMMARY OF THE INVENTION

[0026] According to an aspect of the invention, there is provided a photoblocker usable in additive manufacturing a three-dimensional object, being represented by Formula I:

[0027] Re, R7, Rs, R9 and Rio are each independently hydrogen; and

[0028] Ri, R2, R3, R4 and R5 are each independently selected from hydrogen, hydroxyalkyl and a saccharide moiety.

[0029] According to embodiments of the invention, the saccharide moiety is selected from a monosaccharide and disaccharide moiety.

[0030] According to embodiments of the invention, the saccharide moiety comprises a glucose moiety. According to embodiments of the invention, the R3 is the saccharide moiety.

[0031] According to embodiments of the invention, the Ri is hydrogen.

[0032] According to embodiments of the invention, the R2, R4 and R5 are each hydrogen.

[0033] According to embodiments of the invention, the R2, R4 and R5 are each a hydroxyalkyl.

[0034] According to embodiments of the invention, the hydroxyalkyl is hydroxyethyl.

[0035] According to embodiments of the invention, the R3 is the saccharide moiety; and Ri, R2, R4 and R5 are each hydrogen.

[0036] According to embodiments of the invention, the saccharide moiety is rutinose, the photoblocker being 3-glucoside quercetin.

[0037] According to embodiments of the invention, the R3 is the saccharide moiety; Ri hydrogen; and R2, R4 and R5 are each independently a hydroxyalkyl.

[0038] According to embodiments of the invention, each of R2, R4 and Rs is hydroxyethyl.

[0039] According to embodiments of the invention, the saccharide moiety is rutinose, the photoblocker being troxerutin.

[0040] According to another aspect of the invention, there is provided a photoblocker usable in additive manufacturing a three-dimensional object, the photoblocker being represented by formula I*

[0041] Formula I* wherein

[0042] Rx and Ry are each independently selected from hydrogen (H), alkyl (e.g., lower alkyl as described herein, for example, of 1 to 4 carbon atoms), cycloalkyl and aryl; and Rz is an alkyl (e.g., lower alkyl as described herein, for example, of 1 to 4 carbon atoms), aryl or cycloalkyl.

[0043] According to embodiments of the invention, the photoblocker is HC yellow 9 (HC9).

[0044] According to embodiments of the invention, the photoblocker is characterized by at least one of: biocompatibility and non-toxicity at formulation concentration prior and post curing; solubility of at least 0.05 mg / mL in an aqueous carrier or a curable formulation; absorbance at wavelength from 300 nm to 800 nm; absorbance stability at the wavelength in the presence of a photoinitiator; or chemical stability post irradiation at 240J.

[0045] According to another aspect of the invention, there is provided a curable formulation usable in additive manufacturing a three-dimensional object featuring, in at least a portion thereof, a biological material, the formulation comprising a photoinitiator, a photocurable biological material, a carrier and the photoblocker according to any one of claims 1 to 16.

[0046] According to embodiments of the invention, the curable formulation comprises components as set forth in any one of Tables A-Z, AA or AB.

[0047] According to embodiments of the invention, the curable formulation comprises components as set forth in any one of Tables J-U.

[0048] According to embodiments of the invention, the curable formulation comprises components as set forth in Table K or Table R.

[0049] According to embodiments of the invention, the amount of the photoinitiator in the curable formulation ranges from about 0.1 to 1 % by weight of the total weight of the formulation.

[0050] According to embodiments of the invention, the photoinitiator is an acyl phosphine oxide type photoinitiator.

[0051] According to embodiments of the invention, the acyl phosphine oxide type photoinitiator is 2,4,6-trimethylbenzolydiphenyl phosphine oxide (TMPO) or a salt thereof.

[0052] According to embodiments of the invention, the salt is lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) or sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP).

[0053] According to embodiments of the invention, the amount of the photoblocker in the curable formulation ranges from about 0.02-0.5 % by weight of the total weight of the formulation.

[0054] According to embodiments of the invention, the photocurable biological material comprises a collagen that features a plurality of photocurable groups.

[0055] According to embodiments of the invention, the photocurable groups comprise (meth)acrylic groups.

[0056] According to embodiments of the invention, the collagen is a human Type I collagen.

[0057] According to embodiments of the invention, the collagen is a recombinant collagen.

[0058] According to embodiments of the invention, the collagen is a plant-derived recombinant collagen. According to embodiments of the invention, the collagen is a plant-derived recombinant human Type I collagen.

[0059] According to embodiments of the invention, the amount of the collagen in the curable formulation ranges from about 0.1-2 % by weight of the total weight of the formulation.

[0060] According to embodiments of the invention, the curable formulation further comprises at least one synthetic biocompatible curable material that features a plurality of photocurable groups.

[0061] According to embodiments of the invention, the curable formulation further comprises a photocurable polymeric material.

[0062] According to embodiments of the invention, the curable formulation comprises components as set forth in any one of Tables B-D, F, V and Z.

[0063] According to embodiments of the invention, the curable formulation further comprises at least two photocurable polymeric materials.

[0064] According to embodiments of the invention, the amount of the collagen in the curable formulation ranges from 0.1-1 % by weight of the total weight of the formulation.

[0065] According to embodiments of the invention, at least one of the at least two photocurable polymeric materials has an average molecular weight lower than 10,000 grams / mol (low MW photocurable polymeric material), and at least another one of the at least two photocurable polymeric materials has an average molecular weight higher than 10,000, or higher than 15,000 (high MW photocurable polymeric material), grams / mol; a total amount of the at least two photocurable polymeric materials is in a range of from 1 to 20, or from 5 to 20, or from 5 to 15, or from 7.5 to 15, or from 7.5 to 10, % by weight, of the total weight of the curable formulation; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material ranges from 1: 1 to 1:9, or from 1: 1 to 1:5.

[0066] According to embodiments of the invention, each of the photocurable polymeric material independently is a multifunctional photocurable polymeric material featuring two or more photocurable groups.

[0067] According to embodiments of the invention, the photocurable groups are (meth)acrylic groups.

[0068] According to embodiments of the invention, the (meth)acrylic groups are selected from acrylate and methacrylate groups, provided that at least one of the photocurable polymeric materials features acrylate groups. According to embodiments of the invention, the at least two photocurable polymeric materials features a plurality of acrylate groups and another one of the photocurable polymeric materials features a plurality of methacrylate groups.

[0069] According to embodiments of the invention, the at least one of the photocurable polymeric materials comprises a poly(alkylene glycol) polymeric backbone.

[0070] According to embodiments of the invention, the low MW photocurable polymeric material is a poly(alkylene glycol) that terminates by two (or more) acrylate or methacrylate groups.

[0071] According to embodiments of the invention, the average molecular weight of the low MW photocurable polymeric material ranges from 500 to 10,000, or from 500 to 5,000, or from 500 to 4,000, or from 500 to 3,500, or from 1,000 to 10,000, or from 2,000 to 10,000, or from 1,000 to 8,000, or from 2,000 to 8,000, or from 1,000 to 6,000, or from 2,000 to 6,000, or from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 6,000 or from 3,000 to 5,000 or from 3,000 to 4,000, or from 1,000 to 3,500, or from 500 to 3,500, or from 1,000 to 3,000, or from 1,000 to 2,000, grams / mol.

[0072] According to embodiments of the invention, the high MW photocurable polymeric material comprises a poly(alkylene glycol) polymeric backbone and terminates by two (or more) acrylate or methacrylate groups.

[0073] According to embodiments of the invention, the average molecular weight of the poly (alkylene glycol) polymeric backbone is higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol.

[0074] According to embodiments of the invention, the average molecular weight of the poly (alkylene glycol) polymeric backbone ranges from 10,000 to 40,000, or from 10,000 to 30,000, or from 15,000 to 40,000, or from 15,000 to 30,000, or from 20,000 to 40,000, or from 20,000 to 30,000, or from 20,000 to 25,000, grams / mol.

[0075] According to embodiments of the invention, the high MW photocurable polymeric material is a multi-block (e.g., diblock or triblock) co-polymer that comprises the poly(alkylene glycol) polymeric backbone as one block and at least one additional block that comprises a polymeric backbone of a biodegradable polymer other than poly (alkylene glycol).

[0076] According to embodiments of the invention, the high MW photocurable polymeric material is a tri-block co-polymer that comprises the poly(alkylene glycol) polymeric backbone as a middle block having attached to its termini two additional blocks, each comprising a polymeric backbone of a biodegradable polymer other than poly(alkylene glycol) that terminates by a photocurable group. According to embodiments of the invention, the at least one additional termini block of the biodegradable polymer is a polyester.

[0077] According to embodiments of the invention, the at least one additional termini block of the biodegradable polymer is poly(caprolactones) (PCL).

[0078] According to embodiments of the invention, the molecular weight of the at least one additional termini block is lower than 5,000, or lower than 3,000, or lower than 2,000 grams / mol.

[0079] According to embodiments of the invention, the molecular weight of the at least one additional termini block ranges from 500 to 5,000, or from, 500 to 4,000, or from 500 to 3,000, or from 500 to 2,000, or from 1,000 to 5,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, or from 1,400 to 1,600 grams / mol.

[0080] According to embodiments of the invention, the high MW photocurable polymeric material is a triblock polymeric material that comprises a poly(alkylene glycol) polymeric backbone having a MW higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol and having attached to each of its termini a block of a polyester having a MW lower than 5,000, or lower than 3,000, or lower than 2,000 grams / mol and terminating by at least one (meth)acrylate group.

[0081] According to embodiments of the invention: the low MW photocurable polymeric material is a poly(alkylene glycol) that terminates by two of the acrylate or methacrylate groups and has an average molecular weight that ranges from 1,000 to 10,000, or from 2,000 to 10,000, or from 1,000 to 8,000, or from 2,000 to 8,000, or from 1,000 to 6,000, from 1,000 to 5,000, from 1,000 to 4,000, from 1,000 to 3,000, from 1,000 to 2,000, or from 2,000 to 6,000, or from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 6,000 or from 3,000 to 5,000 or from 3,000 to 4,000 grams / mol; and the high MW photocurable material is a triblock polymeric material that comprises a poly(alkylene glycol) polymeric backbone having a MW higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol and having attached to each of its termini a block of a polyester having a MW lower than 5,000, or lower than 3,000, or lower than 2,000, or lower than 1,600 grams / mol and terminating by the (meth)acrylate group.

[0082] According to embodiments of the invention, the low MW photocurable polymeric material terminate by two acrylate groups.

[0083] According to embodiments of the invention, the high MW photocurable polymeric material terminates by two methacrylate groups.

[0084] According to embodiments of the invention, the weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material is about 1:3. According to embodiments of the invention, the curable formulation comprises components as set forth in any one of Tables E, G, H, I, W, X, Y, AA and AB.

[0085] According to embodiments of the invention, the curable formulation features a viscosity at 22 °C that ranges from 100 to 2,000 mPa- second.

[0086] According to embodiments of the invention, the curable formulation features a viscosity at 22 °C that ranges from 80-3000 centipoises (cP).

[0087] According to embodiments of the invention, the curable formulation features a viscosity at 22 °C that ranges from 70 to 2,500 mPa- second.

[0088] According to embodiments of the invention, the curable formulation features a viscosity at 22 °C that ranges from 200 to 2,500 mPa- second.

[0089] According to embodiments of the invention, the curable formulation features, when hardened a mechanical and / or rheological characteristic as set forth in Table 10.

[0090] According to embodiments of the invention, the curable formulation features, when hardened a tensile strain-at-break of at least about 100% and / or is capable of withstanding a tensile stress of IMPa.

[0091] According to embodiments of the invention, the curable formulation provides, when hardened, a biological or a biocompatible material featuring mechanical properties that meet the requirements of a breast implant.

[0092] According to embodiments of the invention, the curable formulation provides, when hardened, a biological or a biocompatible material featuring at least one of: strain of at least 20 % at 10-100 N; strain of at least 50 % at 100-200 N; compressive strain-at-break higher than 50%; compressive force limit of at least 156N; and volume retention under physiological stress of no more than 30 % strain, at 10N force, as determined in a in a compression force versus strain measurements as described herein.

[0093] According to embodiments of the invention, the carrier is an aqueous carrier.

[0094] According to another aspect of the invention, there is provided a process of additive manufacturing a three-dimensional object featuring, in at least a portion thereof, a biological or a biocompatible material, the process comprising sequentially exposing the curable formulation of any one of claims 17-68, to irradiation at a wavelength of from 300 to 800 nm, in a layerwise manner, and in a configured pattern corresponding to a shape of the object, thereby manufacturing the three-dimensional object. According to embodiments of the invention, the exposing is for time period that ranges from 1 second to 120 or from 1 second to 20 second or from 1 second to 10 second for each layer or is about 12 seconds.

[0095] According to embodiments of the invention, the irradiation is at a wavelength that ranges from 300 to 600 nm, or from 300 to 500 nm, or from 350 to 450 nm, or is 385 nm.

[0096] According to embodiments of the invention, the irradiation is at a power intensity level that ranges from 1 to 150 mW / cm2or from 1 to 50 mW / cm2or is about 10mW / cm2.

[0097] According to embodiments of the invention, the irradiation is at an energy level that ranges from 1 to 250 mJ / cm2or from 1 to 210 mJ / cm2or from 1 to 130mJ / cm2or is about 120mJ / cm2.

[0098] According to embodiments of the invention, the additive manufacturing is DLP.

[0099] According to an aspect of the invention there is provided a kit comprising the photoblocker of any one of claims 1-16, a photoinitiator and a photocurable biological material.

[0100] According to further embodiments, the kit comprises components as set forth in Table K and Table R.

[0101] According to further embodiments, the kit further comprises a polymer.

[0102] According to further embodiments, the photoinitiator is an acyl phosphine oxide type photoinitiator.

[0103] According to further embodiments, the acyl phosphine oxide type photoinitiator is 2,4,6- trimethylbenzolydiphenyl phosphine oxide (TMPO) or a salt thereof.

[0104] According to further embodiments, the salt is lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) or sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP).

[0105] According to further embodiments, the photocurable biological material comprises a collagen that features a plurality of photocurable groups.

[0106] According to further embodiments, the collagen is lyophilized.

[0107] According to further embodiments, the photocurable groups comprise (meth)acrylic groups.

[0108] According to further embodiments, the collagen is wherein the collagen is a plant-derived recombinant human Type I collagen.

[0109] According to further embodiments, the degree of collagen methacrylation is at least 37 %.

[0110] According to further embodiments, the degree of collagen methacrylation is at least 50 %.

[0111] According to further embodiments, the degree of collagen methacrylation is at least 90 %.

[0112] 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 the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0113] Implementation of the method and / or system of embodiments of the invention can involve performing or completing selected tasks manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of embodiments of the method and / or system of the invention, several selected tasks could be implemented by hardware, by software or by firmware or by a combination thereof using an operating system.

[0114] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0115] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0116] FIGs. 1A-B presents the UV absorbance spectra of 10 mM HC1 or DDW solutions containing 10 mM minocycline and other tested exemplary photoblocker candidates (at a final concentration of 0.1 or 10 mM). FIG. 1A presents comparative plots of normalized absorbance spectra of exemplary insoluble versus soluble tested photoblocker candidates. FIG. IB further presents the UV absorbance spectra of exemplary soluble tested photoblocker candidates (Minocycline, quinoline yellow, PB-7 and PB-9) prior to and post irradiation at 365 nm for 20 seconds at 100% intensity.

[0117] FIG. 2 A presents photographs of vials containing 10 mM HC1 or DDW solutions containing 0.1 mM or ImM minocycline HC1 (PB-1) (hereinafter also referred to interchangeably as minocycline or MC), quinoline yellow (QY; PB-2) or other exemplary photoblocker candidates (PB numbers shown on Figure) and NAP (0.5 % by weight), before (upper panel) and after (lower panel) exposure to irradiation at 365 nm for 20 seconds at 100% intensity.

[0118] FIG. 2B presents comparative plots of the UV absorbance spectra of exemplary solutions containing 4-nitroaniline (PB-7) and mordant yellow 10 (PB-9) which were found unstable post UV irradiation versus exemplary solutions containing minocycline HC1 (PB-1) and Quinoline Yellow (PB-2) which were found to be stable post UV irradiation.

[0119] FIG. 2C presents comparative plots of UV absorbance spectra of solutions containing HC Yellow 9 (PB-13) (hereinafter referred to interchangeably as HC9 yellow or HC9) and Chlorophyllin (PB-14) provided as exemplary dye substance candidates which were found stable post UV irradiation at 365nm 100% 20sec in the presence NAP.

[0120] FIG. 3 A presents the absorbance spectra of a solution containing PB-17 (rutin 0.2 mM; upper curve (black)) and PB-19 (curcumin 0.27 mM; lower curve - grey).

[0121] FIG. 3B presents the absorbance spectra of solutions containing 0.2 mM PB-17, NAP, and 0.2 mM PB-17 and NAP, before and after irradiation at 385 nm.

[0122] FIGs. 4A-B present pictorial representations of the XY plane (FIG. 4 A) and Z axis (FIG. 4B) of a calibration model as shown in FIG. 6 A printed with curable formulation comprising PB- 17 (0.004% by weight), methacrylated rh-Collagen (CMR; 5 mg / mL), 15%PEGDA3.4K, NAP (0.5 % by weight).

[0123] FIG. 5 presents plots showing the kinetics rate (time to reach storage modulus (G’)) of formulation H5.

[0124] FIGs. 6Aa-6Ac present an isometric view (6Aa), the XY plane (6Ab) and the Z axis (6Ac) of a calibration model used in printing resolution studies.

[0125] FIG. 6Ad presents a drawing of both views of a unit cell (UC) model used to ensure sufficient resolution is kept in XY (left) and Z (right) while printing a representative part of the SC breast scaffold.

[0126] FIG. 6B presents pictorial representations of the XY plane (left) and Z axis (right) of a calibration model printed with curable formulation H4 at 35 mJ / cm2.

[0127] FIGs. 6C and 6D present pictorial representations of the XY plane (left) and Z axis (right) of calibration models printed with curable formulations H8 and H9 at 80 mJ / cm2respectively.

[0128] FIGs. 6E and F present pictorial representations of calibration models printed with curable formulation H10. FIG 6E depicts the XY plane (left) printed at 35 mJ / cm2and FIG. 6F depicts the XY plane (left) and Z axis (right) of a calibration model printed at 120 mJ / cm2

[0129] FIG. 6G presents pictorial representations of the XY plane (left) and Z axis (right) of calibration model printed with curable formulation H13 at 110 mJ / cm2.

[0130] FIG. 6H presents pictorial representations of the XY plane (left) and Z axis (right) of calibration model printed with curable formulation H12 at 200 mJ / cm2.

[0131] FIG. 61 presents pictorial representations of the XY plane (left) of calibration model printed with curable formulation Hl 1 at 400 mJ / cm2.

[0132] FIG. 6J presents pictorial representations of the XY plane and Z axis of calibration models and UC models printed with curable formulation H15 at 220 mJ / cm2versus H22 printed at 160- 200 mJ / cm2 FIG. 6K presents pictorial representations of different 3D models printed with curable formulation H22.

[0133] FIG. 6L presents a comparative data of the mechanical properties of dog bone models printed with H15 versus H22(left) and dimensions of such dogbones (right).

[0134] FIG. 6M presents comparative data of the mechanical properties of cylinders printed with H15 versus H22 (left) and dimensions of such cylinders (right).

[0135] FIG. 6N presents bar graphs of two independent experiments of nHDF proliferation (day 4, 8,11 and day 4,7 ,11) on H22 meshes.

[0136] FIG. 60 presents photographs of nHDF seeded on H22 meshes.

[0137] FIGs. 7A-D present comparative data obtained using Instron machine, showing the mechanical properties of dogbone models printed with a curable formulation that comprises PB- 13 (H6), compared to the same formulation (F0) comprising minocycline as a photoblocker.

[0138] FIG. 8 presents a bar graph showing cell viability upon incubating cell seeded on a model made of a curable formulation that comprises MC (F01) or PB-13 (H7) for 15 days.

[0139] FIG. 9 presents the absorbance spectra of a solution containing 3-P-D-Glucosylquercetin (PB23) (10 mM solution in 10 mM HC1), NAP in DDW (25mg / ml and 0.5 w / w%), 3- -D- Glucosylquercetin (10 mM solution in 10 mM HC1) and NAP (0.5 w / w%), before and post irradiation at 385nm lOsec.

[0140] FIGs. 10A-B present plots showing the kinetics rate (time to reach storage modulus (G’)) of curable formulations F24 and F26 respectively).

[0141] FIG. 11A (a) and (b) present pictorial representations of the XY plane and Z axis respectively of a calibration model printed with curable formulation A printed with an energy of 110 mJ / cm2.

[0142] FIGs. 1 IB and 11C present pictorial representations of the XY plane and Z axis respectively of a calibration model printed with curable formulations F24 or F26 respectively at printing energy of 120 mJ / cm2(a, b).

[0143] FIGs. 12Aa-12Ad, 12Ba-12Bd and 12Ca-12Cd present comparative data of compression test, which demonstrate the mechanical properties of 20 cc scaffolds printed with a curable formulation A (Figures 12Aa, 12Ab, 12Ac, 12Ad), F24 (12Ba, 12Bb, 12Bc, 12Bd), F26 (12Ca, 12Cb, 12Cc, 12Cd) respectively that comprises 3-P-D-Glucosylquercetin compared to the same formulation Fl, F13, or F18 comprising 0.2 w / w% minocycline respectively.

[0144] FIG. 13 presents the absorbance spectra at 385 nm of a solution containing 10 mM solution Troxerutin (PB22), Troxerutin (10 mM solution in 10 mM HC1) and NAP (0.5 w / w%), before and after irradiation at 385nm lOsec. FIGs. 14A-B present plots showing the kinetic rate of formulations F23 or F25 respectively.

[0145] FIGs. 15 A-B (a)&(b) present pictorial representations of the XY plane and Z axis respectively of a calibration model printed with curable formulations AA or F23 respectively printed with energy of 150 / cm2.

[0146] FIG. 15C(a)&(b) presents pictorial representations of the XY plane and Z axis respectively of a calibration model printed with curable formulation F25 printed with energy of 140 mJ / cm2.

[0147] FIGs. 16A-M present comparative data of the mechanical properties of dog bone models, cylinders models, and 20cc scaffolds printed with curable formulation F23 (FIG. 16A-C) or F25 (FIG. 16D, G and J) that comprises Troxerutin compared to the same formulations F13 and F18 respectively comprising minocycline. FIG. 16A and FIG. 16D Material tensile test, FIG. 16B and FIG. 16G Material compression test and FIG.16C and FIG.16J 20cc lattice compression test of formulations F23 and F25 respectively. FIGs. 16E, 16H and 16K material tensile tests, material compression tests and lattice compression test respectively comparing mechanical properties “as print” of dogbones, cylinders and 20cc lattice printed from three different batches of F25. FIGs. 16F and 161 material tensile and compression tests of F25 dogbones and cylinders respectively comparing mechanical properties of “as print” and post-process. FIG. 16L lattice compression test comparing mechanical properties of F2540% and 50% vf lOOcc implants post process printed from the same F25 batch. FIG. 16M lattice compression test comparing mechanical properties of F25 50% vf VBHB lOOcc implants prior and post process.

[0148] FIG. 17 presents comparative data of mechanical properties of 20cc scaffolds printed with curable formulations F24, F26 (both GQ-based) F23, F25 (both TR-based) compared to the comparable formulations F13 and Fl 8 comprising 0.2% minocycline.

[0149] FIG. 18 is a comparative bar graph showing cell viability upon incubating nHDF cell line seeded on a model made of a curable formulation that comprises GQ (formulation F24) or TR (formulation F23) compared to control (plastic).

[0150] FIG. 19 are photographic representations of FIG. 18.

[0151] FIG. 20 is a bar graph illustrating nHDF cell proliferation with conditional medium. 50,000 cells were seeded on cell culture plate and incubated with printed models conditioned medium. DMEM medium - medium that was incubated in the cell culture incubator for three days. The control group received fresh medium. Cell proliferation was measured using the Presto Blue assay.

[0152] FIGs. 21 A-B are bar graphs showing the effect of PBs (GQ or TR) at formulation concentration on nHDF (FIG. 21 A) and L929 (FIG. 2 IB) viability. 10,000 cells were seeded on a cell culture plate and incubated for 24 hours. After this period, the cells were exposed to a medium containing PBs. Cell viability was then normalized to that of the control cells. FIGs. 21C-D are bar graphs nHDF and L929 cell proliferation on F25 or F23 printed models: A total of 50,000 cells were seeded onto printed plates and incubated for 7 days. Cell proliferation was measured using the Presto Blue assay, and cell numbers were calculated based on a calibration curve. The average cell count from six models is presented.

[0153] FIG. 22 is a bar graph illustrating the mass loss after 2 (not shown) and 7 days for scaffolds samples printed from Formulations F24, F23 and F13 in biocarbonate / carbonate buffer (pH 10) in comparison to control samples in PBS xl (pH 7) at 37 °C.

[0154] FIG. 23 is a bar graph illustrating the mass loss after 2- and 7-days comparing mass loss of formulations F13 and Fl 8 in biocarbonate / carbonate buffer (pH 10) in comparison to control samples in PBSxl(pH 7) at 37 °C.

[0155] FIG. 24A presents graphs comparing degradation rate of samples printed from F23 and F25 in biocarbonate / carbonate buffer (pH 10) at 37 °C until fully degraded compared to control samples in PBS (pH 7) at 37 °C after 80 days.

[0156] FIG. 24B presents graphs comparing degradation rate of samples printed from F23 and F25 in 6% H2O2 at 37 °C (pH 6) until fully degraded compared to control samples in PBS (pH 7) at 37 °C after 50 days.

[0157] FIG. 24C presents graphs comparing the degradation rate of samples printed from F23 and F25 subjected to identical real-time degradation conditions under physiological temperature and pH (37°C pH 7) and humidity over at 3, 6 and 9 months.

[0158] FIG. 24D is a representative image of samples printed from F23 and F25 subjected to identical real-time degradation conditions under physiological temperature and pH (37 °C pH 7) and humidity over a 9-month period.

[0159] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0160] The present invention, in some embodiments thereof, relates to additive manufacturing, and more particularly, but not exclusively, to light-absorbing substances that can be beneficially included in formulations usable in additive manufacturing of 3D objects featuring a biological material in at least a portion thereof.

[0161] Before explaining 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 set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Currently practiced additive manufacturing (AM) methodologies (including digital light processing bioprinting) oftentimes require the use of a photoblocker (PB) for achieving improved performance of both the process and the obtained three-dimensional object.

[0162] The present inventors have now identified particular chemical agents that can be beneficially used as photoblockers in 3D bioprinting of 3D objects that feature a biological material as defined herein in at least a portion thereof.

[0163] As demonstrated in the Examples section that follows, Applicants tested a myriad of dyes and chemical agents for properties deemed important for effective use (e.g., solubility, toxicity, stability, biocompatibility, absorbance at light at a wavelength suitable for curing (hardening) a curable formulation).

[0164] It was found that several PB candidates whilst being non-toxic, water soluble and featuring absorbance at the desired UV-vis range did not perform when used in AM, since under the process condition (e.g. irradiation in the presence of a selected photoinitiator) these candidates underwent decomposition or otherwise a chemical change that resulted in a loss of absorbance (shift or attenuation) at the indicated wavelength (FIGs. 1A-2B).

[0165] The present inventors uncovered three dyes which presented with high optical activity at 385 nm, (in the presence and absence of photoinitiator) and which retained their absorbance during AM. Two of these having a flavanol structure - 3-P-D-Glucosylquercetin (GQ) and Troxerutin (TR) and the other being HC yellow no. 9 (HC9). Each of these dyes when used in curable formulations generated clear and transparent implants. These candidates were also found to meet the requirements of solubility, kinetics, toxicity and biocompatibility making them promising PB candidates in the process of AM.

[0166] As described and demonstrated in the Examples section that follows, the present inventors have studied the compatibility and suitability of these three PBs in terms of the process requirements, and have surprisingly uncovered that all three feature the required solubility in the curable formulation, the required UV absorbance profile which remains substantially unchanged in the presence of NAP also following irradiation at 365 / 385 nm (FIG. 2C, FIG.9, FIG.13), the required chemical stability which is maintained post irradiation at high intensity and, at the same time, do not adversely affect homogeneity or the solubility of other components in a curable formulation comprising the same, nor the curing kinetics (FIG. 5, FIG.10 and FIG.14).

[0167] Curable formulations which were particularly effective for high resolution AM were developed (FIG. 6B-D, FIG. 6G, FIG. 6J, FIG. 6K, FIG.11 and FIG.15) and the newly discovered PBs were found to be compatible therewith. The biological (FIG. 60 FIG.8, FIGs. 18-21), mechanical (FIGs 6E&M, FIG. 7, FIG.12, FIGs.16- 17), rheological properties (FIG. 5, FIG.10 and FIG.14) and degradation profiles (FIGs. 22-24) of such formulations were also characterized. Such formulations are particularly useful for AM of objects which require high resolution and biocompatibility.

[0168] As is illustrated herein under and in the examples section, which follows, the present inventors showed that HC9 was a suitable PB candidate with respect to resolution (FIGs. 6B,6J, 6K) and mechanical properties (FIGs. 6, 7A-D). In addition, HC9 did not substantially impact viability of nHDF and L929 cell at formulation concentration (FIGs. 60 and FIG. 8).

[0169] Whilst further reducing the present invention to practice, the present inventors compared dogbones, cylinders or scaffolds printed using curable formulations comprising the newly discovered PBs and compared them with those printed using curable formulations comprising minocycline.

[0170] The present inventors showed that GQ-based scaffolds printed from formulations comprising 15%PEGDA3.4K were of a higher strength and stiffness compared to scaffolds printed from the same formulations using Minocycline as the PB (FIG. 12Aa-d). GQ-based scaffolds printed from formulations comprising a combination of a short chain polymer and a long chain polymer (at a ratio of 3: 1) were less stiff than minocycline-based scaffolds but of comparable strength (FIGs. 12B-Ca-d). In addition, GQ did not substantially impact viability of nHDF cells (FIG. 18) and L929 cells (not shown) at formulation concentration. Furthermore, the present inventors showed that GQ-based scaffolds showed satisfactory resolution in the XY plane and Z axis (FIGs. 11A-C).

[0171] Whilst further reducing the present invention to practice, the present inventors showed that TR-based scaffolds printed from formulations comprising a combination of a short chain polymer and a long chain polymer (at a ratio of 3: 1) were less stiff than minocycline-based scaffolds but of comparable strength (FIG. 16J). In addition, TR did not substantially impact viability of nHDF cells (FIG. 18) and L929 cells (not shown) at formulation concentration. Cell viability was not significantly affected when cells were incubated with TR for 72 hours and both F23 and F25 formulations supported the growth and proliferation of both nHDF and L929 cells on printed models therefrom at 7 days (FIGs. 19-21). In addition, the present inventors showed that TR-based scaffolds showed satisfactory resolution in the XY plane and Z axis (FIGs. 15A-C).

[0172] The present inventors demonstrated that for additive manufacturing of breast implants the formulation should ideally following photocuring and washing (“as print”) and optionally post process) a hardened material which exhibits tensile strain-at-break of at least 70 % or even at least 100% and tensile strength of at least 0.7 MPa or even at least IMPa (e.g., when measured as described herein for standard models such as dogbone models as described, obtained by additive manufacturing as described herein). The stiffness of such dogbones could vary according to the manufacturing and post-printing processes, allowing for the design of different applications. For example, at 70 % strain, the material exhibits tensile stress of 0.7 MPa, or of 0.75 MPa, or of 0.8 MPa, or higher. For example, at 100 % strain, the material exhibits tensile stress of 0.7 MPa, or of 0.75 MPa, or of 0.8 MPa, or of 0.85 MPa, or of 0.9 MPa, or of 0.95 MPa, or of 1.00 MPa, or higher. For example, at 110 % strain, the material exhibits tensile stress of 0.7 MPa, or of 0.75 MPa, or of 0.8 MPa, or of 0.85 MPa, or of 0.9 MPa, or of 0.95 MPa, or of 1.00 MPa, or of 1.10 MPa, or of 1.15 MPa, or of 1.20 MPa, or higher. (FIGs. 16E) All when measured as described herein.

[0173] Whilst reducing the present invention to practice, the present inventors uncovered that scaffolds fabricated from a combination of short chain curable polymer and long chain curable polymer PEG-PCL 23k degrade significantly faster than those printed with the same formulation other than long chain curable polymer PEG 20k under the same real time conditions (i.e., pH 7 temperature of 37°C and humidity). The degradation observed in PEG-PCL 23k is attributed to the hydrolytically labile PCL block, in contrast to the higher stability of PEG 20k. The study results after a 9-month period in vitro emphasize the critical role of polymer composition in designing biodegradable scaffolds and the importance of tailoring degradation rates to synchronize specific regenerative requirements. The fast degradation mechanism of the PEG PCL may be prone to limit this synchronization while PEG provides longer stability periods. This study is aligned with the conclusions of accelerate degradations study wherein PEG 20K DMA exhibited relatively higher resistance to degradation in pH 10, compared to PEG PCL 23 K. The PEG PCL 23 K combines two degradation mechanisms. The main mechanism is via hydrolysis of methacrylate ester while the ether PEG chain undergoes oxidation slowly over time (FIGs. 22-24).

[0174] The present inventors also found that the structural design of the implant and post process steps affects both mechanical (FIGS. 16L&M) and biological properties and as described in US Application 63 / 696,908 and co-filed PCT having title “Formulations and designs for degradable soft tissue implants”, having a same applicant and having docket number 104970, which this application claims priority therefrom.

[0175] Embodiments of the present invention therefore relate to the newly uncovered PBs HC9, TR, GQ and / or other compounds featuring a quercetin skeleton suitable for use in curable formulations (also referred to herein as “modeling material formulations” or “bioink compositions”), and to additive manufacturing processes utilizing same.

[0176] According to some embodiments, exemplary photoblockers that are usable in the context of the present embodiments can be collectively represented by Formula I:

[0177]

[0178] Formula I wherein:

[0179] Re, R7, Rs, R9 and Rio are each independently hydrogen; and

[0180] Ri, R2, R3, R4 and R5 are each independently selected from hydrogen, hydroxyalkyl and a saccharide moiety.

[0181] The term “saccharide” as used herein encompasses monosaccharides, disaccharides and oligosaccharides. The term "monosaccharide", as used herein and is well known in the art, describes a simple form of a sugar that consists of a single saccharide molecule, which can be open-chain or cyclic (e.g., pyranose- or furanose-based), and which cannot be further decomposed by hydrolysis. Most common examples of monosaccharides include glucose (dextrose), fructose, galactose, and ribose. Monosaccharides can be classified according to the number of carbon atoms of the carbohydrate, i.e., triose, having 3 carbon atoms such as glyceraldehyde and dihydroxyacetone; tetrose, having 4 carbon atoms such as erythrose, threose and erythrulose; pentose, having 5 carbon atoms such as arabinose, lyxose, ribose, xylose, ribulose and xylulose; hexose, having 6 carbon atoms such as allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, rhamnose and tagatose; heptose, having 7 carbon atoms such as mannoheptulose, sedoheptulose; octose, having 8 carbon atoms such as 2-keto-3-deoxy-manno- octonate; nonose, having 9 carbon atoms such as sialose; and decose, having 10 carbon atoms. Monosaccharides are the building blocks of oligosaccharides and disaccharides like sucrose (common sugar).

[0182] The term “disaccharide” describes a compound two monosaccharide units, which can be the same or different, covalently bound to one another, typically via a glucosyl bond. Examples include sucrose and maltose. The term “oligosaccharide” as used herein describes a compound that comprises three or more monosaccharide units, as these are defined herein, which can be the same or different. Preferably, the oligosaccharide comprises 3-6 monosaccharides units.

[0183] According to some of any of the embodiments described herein for Formula I, the saccharide moiety is a monosaccharide moiety or a disaccharide moiety.

[0184] According to some of any of the embodiments described herein for Formula I, the saccharide moiety, whether a monosaccharide or a disaccharide, comprises a glucose moiety.

[0185] According to some of any of the embodiments described herein for Formula I, the saccharide moiety is a disaccharide moiety.

[0186] According to some of any of the embodiments described herein for Formula I, the saccharide moiety is a disaccharide moiety and one of the saccharides is glucose.

[0187] According to some of any of the embodiments described herein for Formula I, the saccharide moiety is a disaccharide moiety that comprises glucose and rhamnose.

[0188] According to some of any of the embodiments described herein for Formula I, the saccharide moiety is rutinose: wherein the dashed line represents the attachment point to the respective oxygen in Formula I.

[0189] According to some of any of the embodiments described herein for Formula I, R3 is the saccharide moiety, as described herein in any of the respective embodiments.

[0190] According to some of any of the embodiments described herein for Formula I, R3 is a disaccharide moiety that comprises glucose.

[0191] According to some of any of the embodiments described herein for Formula I, R3 is rutinose.

[0192] According to some of any of the embodiments described herein for Formula I, Ri is hydrogen. According to some of any of the embodiments described herein for Formula I, R2, R4 and R5 are each hydrogen.

[0193] According to some of any of the embodiments described herein for Formula I, Ri, R2, R4 and R5 are each hydrogen and R3 is a saccharide moiety as described herein. According to some of these embodiments, R3 is a disaccharide moiety that comprises glucose.

[0194] According to some of these embodiments, R3 is rutinose, and the photoblocker is 3- glucoside quercetin:

[0195] 3-glucoside quercetin.

[0196] According to some of any of the embodiments described herein for Formula I, at least one of Ri, R2, R4 and R5 is hydroxyalkyl.

[0197] Herein and in the art, the term “alkyl” describes any saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., “1 to 20”, is stated herein, it implies that the group, in this case the hydrocarbon, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. Preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or non-substituted.

[0198] Herein, a “hydroxyalkyl” describes an alkyl, as defined herein, preferably a lower alkyl of 1 to 4 carbon atoms, which is substituted by one or more hydroxy (-OH) substituents. Preferably, the term “hydroxyalkyl” refers to an alkyl that is terminated by a hydroxy substituent, that is, the hydroxy substituent is a substituent of a terminal carbon atom of the alkyl substituent.

[0199] In some embodiments, the hydroxyalkyl comprises one hydroxy substituent on the terminal carbon atom of the alkyl, and the alkyl is otherwise not substituted.

[0200] In some embodiments, the hydroxyalkyl is hydroxyethyl, -CH2-CH2-OH. Alternatively, the hydroxyalkyl can be hydroxymethyl, hydroxybutyl or hydroxypropyl.

[0201] According to some of any of the embodiments described herein for Formula I, at least two or at least three of Ri, R2, R4 and R5 are each independently a hydroxyalkyl. According to some of any of the embodiments described herein for Formula I, at least two or at least three of Ri, R2, R4 and R5 are each hydroxyethyl.

[0202] According to some of any of the embodiments described herein for Formula I, Ri is hydrogen and one, two or all of R2, R4 and R5 are each a hydroxyalkyl such as hydroxyethyl. According to some of any of the embodiments described herein for Formula I, Ri is hydrogen and each of R2, R4 and R5 is independently a hydroxyalkyl such as hydroxyethyl.

[0203] According to some of any of the embodiments described herein for Formula I, Ri is hydrogen and each of R2, R4 and R5 is hydroxyethyl. According to some of these embodiments, R3 is a saccharide moiety as described herein. According to some of these embodiments, R3 is a disaccharide moiety that comprises glucose. According to some of these embodiments, R3 is rutinose, and the photoblocker is troxerutin.

[0204] Troxerutin.

[0205] According to some embodiments, the photoblocker is an analog of HC yellow 9, featuring a structure or a salt thereof (e.g., HC1 salt) represented by Formula I*:

[0206] Formula I* Wherein Rx and Ry are each independently selected from hydrogen (H), alkyl (e.g., lower alkyl as described herein, for example, of 1 to 4 carbon atoms), cycloalkyl and aryl; and Rz is an alkyl (e.g., lower alkyl as described herein, for example, of 1 to 4 carbon atoms), aryl or cycloalkyl.

[0207] According to some embodiments, the photoblocker is HC yellow 9, featuring the following structure or a salt thereof (e.g., HC1 salt).

[0208] HC yellow 9.

[0209] The photoblockers described herein, are capable of absorbing light at a wavelength suitable for curing (hardening) a curable formulation of interest.

[0210] According to some of any of the embodiments described herein, the photoblockers described herein are capable of absorbing light at a wavelength at the UV-visual range (e.g. in a range of from 300 nm to 800 nm, or from 300 nm to 600 nm, or from 300 nm to 500 nm, or from 350 nm to 450 nm, or, for example, at 385 or at 405 nm).

[0211] In one embodiment, the photoblocker features an absorbance at the wavelength at which the curable formulation is irradiated and subsequently crosslinks or hardens.

[0212] Preferably, the absorbance does not significantly change in the presence of a selected photoinitiator (as further described herein below) following irradiation at the selected wavelength.

[0213] According to some embodiments, a significant change is a change of no more than 50 %, no more than 40 %, no more than 30 %, no more than 25 %, no more than 20 %, no more than 15 %, no more than 10 %, no more than 5 %, in absorbance following the irradiation.

[0214] In still another embodiment, the photoblockers described herein are soluble in an aqueous carrier. According to some of any of the embodiments, the photoblockers are water-soluble, as defined herein. According to some of any of the embodiments, the photblockers are water- miscible, as defined herein. Herein throughout, the term “water-miscible” describes a material which is at least partially dissolvable or dispersible in water, that is, at least 50 % of the molecules move into the water upon mixture. This term encompasses the terms “water-soluble” and “water dispersible”.

[0215] Herein throughout, the term “water-soluble” describes a material that when mixed with water in equal volumes or weights, a homogeneous solution is formed.

[0216] For example, the photoblockers described herein feature solubility of at least 0.05, or at least 1, or at least 10, mg / mL in water and / or in the selected carrier.

[0217] The photoblockers described herein are biocompatible (e.g., non-toxic, preferably recognized as safe for use by animated subjects).

[0218] According to a particular embodiment, the photoblocker is Troxerutin (TR).

[0219] TR is commercially available from a variety of sources including, but not limited to ChemSene, Angene (e.g. Lot No. AG00397y), Merk and AA Blocks (e.g., Cat. No.:AA00IC16).

[0220] According to a particular embodiment, the photoblocker is 3-P-D-Glucosylquercetin (GQ).

[0221] GQ is commercially available from a variety of sources including, but not limited to Angene (e.g. Lot No. AGN23-14582181), Merk and AA Blocks.

[0222] HC9 is commercially available from a variety of sources including, but not limited to Merk, ChemSene and Angene.

[0223] The photoblockers described herein may be used for a myriad of purposes including the process of AM (e.g., digital light processing bioprinting), as described in further detail hereinafter.

[0224] Thus, according to another aspect of the invention there is provided a curable formulation, comprises one or more photocurable materials, a photoinitiator a photoblocker as described herein.

[0225] It will be appreciated that the above mentioned components may optionally be divided between two or more modeling material formulations, as long as the additive manufacturing requirements are met.

[0226] As used herein, the term “curable” describes a formulation or a material that is capable of undergoing curing, or hardening (e.g., a change in viscosity or in storage modulus (G’ )), as defined herein, when exposed to a suitable curing condition.

[0227] According to some of any of the embodiments described herein, the curable formulation features, when cured or hardened, storage modulus (G’) that ranges from about 100 Pa to about 50,000 Pa, or from about 1,000 Pa to about 50,000 Pa, or from about 100 Pa to about 40,000 Pa, or from about 1,000 Pa to about 40,000 Pa, or from about 100 Pa to about 30,000 Pa, or from about 1,000 Pa to about 30,000 Pa, or from about 100 Pa to about 25,000 Pa, or from about 1,000 Pa to about 25,000 Pa, or from about 1,000 Pa to about 20,000 Pa, or from about 100 Pa to about 20,000 Pa, or from about 1,000 Pa to about 20,000 Pa, or from about 5,000 Pa to about 30,000 Pa, or from about 5,000 Pa to about 25,000 Pa, or from about 10,000 Pa to about 30,000 Pa or from about 10,000 Pa to about 25,000 Pa, including any intermediate values and subranges therebetween. According to a preferred embodiments described herein, the curable formulation features, when cured or hardened, storage modulus (G’) that ranges from about from about 7170Pa to about 24,770Pa, from 8,000 Pa to about 20,000 Pa, or from about 4,240 Pa to about 5,020 Pa.

[0228] According to some of any of the embodiments described herein, the photoblocker is Troxerutin and its amount ranges from 0.01 to 5 %, or from 0.01 to 2 %, or from 0.01 to 1 %, or from 0.05 to 1.5 %, 0.01 to 0.5 %, or from 0.01 to 0.4 %, or from 0.01 to 0.3%, from 0.01 to 0.2%, from 0.01 to 0.1% or from 0.05 to 1.5 %, 0.1 or from 0.1 to 1 %, or from 0.1 to 1.5 %, 0.1 to 0.5 %, or from 0. 1 to 0.4 %, or from 0.1 to 0.3%, from 0.1 to 0.2%, from 0.1 to 1% or from 0.05 to 1.5 %, by weight of the total weight of the formulation or composition comprising same, including any intermediate values and subranges therebetween.

[0229] According to some of any of the embodiments described herein, the photoblocker is P-D- Glucosylquercetin and its amount ranges from 0.01 to 5 %, or from 0.01 to 2 %, or from 0.01 to 1 %, or from 0.05 to 1.5 %, 0.01 to 0.5 %, or from 0.01 to 0.4 %, or from 0.01 to 0.3%, from 0.01 to 0.2%, from 0.01 to 0.1%or from 0.05 to 1.5 %,0.1 or from 0.1 to 1 %, or from 0.1 to 1.5 %, 0.1 to 0.5 %, or from 0. 1 to 0.4 %, or from 0.1 to 0.3%, from 0.1 to 0.2%, from 0.1 to l%or from 0.05 to 1.5 %, by weight of the total weight of the formulation or composition comprising same, including any intermediate values and subranges therebetween.

[0230] According to some of any of the embodiments described herein, the photoblocker is HC9 and its amount ranges from 0.001 to 5 %, or from 0.001 to 2 %, or from 0.001 to 1 %, or from 0.005 to 1.5 %, 0.001 to 0.5 %, or from 0.001 to 0.4 %, or from 0.001 to 0.3%, from 0.001 to 0.2%, from 0.001 to 0.1%or from 0.005 to 1.5 %,or from 0.01 to 1 %, or from 0.01 to 1.5 %, 0.01 to 0.5 %, or from 0.01 to 0.4 %, or from 0.01 to 0.3%, from 0.01 to 0.2%, from 0.01 to 1% or from 0.01 to 0.1 %, or from 0.01 to 0.5 %, 0.01 to 0.05 %, or from 0.01 to 0.04 %, or from 0.01 to 0.03%, from 0.01 to 0.02%, from 0.01 to 0.01% from 0.01 to 0.025 %,by weight of the total weight of the formulation or composition comprising same, including any intermediate values and subranges therebetween.

[0231] According to some of any of the embodiments described herein, the curable formulation features a pH that ranges 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 values and subranges therebetween. .

[0232] According to some of any of the embodiments described herein, the curable formulation features a pH that ranges from about 2 to about 7, or from about 2 to about 6, or from about 2 to about 5, or from about 2 to about 4, or from about 2 to about 3, or from about 2.4 to about 3, including any intermediate values and subranges therebetween.

[0233] According to some of any of the embodiments described herein, the curable formulation features a pH that ranges from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, from about 1 to about 2, from about 2 to about 5, from about 2 to about 4, from about 2 to about 3, from about 2 to about 3.5 or from about 2.5 to about 3.5 including any intermediate values and subranges therebetween. In a preferred embodiment the curable formulation features a pH that ranges between 2.4-3.0.

[0234] According to some of any of the embodiments described herein, the curable formulation features a shear-thinning behavior (e.g., at room temperature, for example, from 20 to 25 °C) and is a shear-thinning composition.

[0235] The term “shear-thinning” describes a property of a fluidic material that is reflected by a decrease in its viscosity (increase in its fluidity) upon application of shear forces (under shear strain), at an indicated temperature, when determined using a rheometer as described in the Examples section that follows.

[0236] In some of the present embodiments, a shear-thinning material is such that exhibits a significant, e.g., at least 100 %, reduction in its shear modulus upon increasing the shear strain from about 1% to above 50 %. Shear-thinning materials therefore exhibit a shear-dependent viscosity profile.

[0237] According to some of any of the embodiments described herein, the curable formulation features a fast recovery rate upon a change in the applied shear force (a fast shear recovery).

[0238] According to some of any of the embodiments described herein, the curable formulation features a change of no more than 6 %, or of no more than 10 % upon shear rest (zero shear force) of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes and even 10 minutes.

[0239] According to some of any of the embodiments described herein, the curable formulation features at least 80 % recovery, or at least 85 % recovery, or at least 90 % recovery, or at least 92 % recovery, of its viscosity upon increasing the shear rate from about 0 1 / sec or 1 1 / sec to above 50 1 / sec, for a time period of at least 1 minute (e.g., from about 60 seconds to about 120 seconds, e.g., about 100 seconds). According to some of any of the embodiments described herein, the curable formulation features a viscosity of no more than 100 centipoises (cP), no more than 200 cP, or no more than 250 cP, no more than 300 cP, no more than 1000 cP at a shear rate of 10 1 / sec, at room temperature, as described herein, when determined using a rheometer as described in the Examples section that follows.

[0240] According to some embodiments of the invention, the curable formulation features a viscosity at room temperature, at a shear rate of 10 1 / sec, that ranges from 80 to 3,000 cP. According to some embodiments of the invention, the curable formulation features a viscosity at room temperature, at a shear rate of 10 1 / sec, that ranges from 80 to 250 cP. According to some embodiments of the invention, the curable formulation features a viscosity at room temperature, at a shear rate of 10 1 / sec, that ranges from 720 to 3000 cP. According to some embodiments of the invention, the curable formulation features a viscosity at a room temperature, at a shear rate of 10 1 / sec, that ranges from 250 to 3000 cP. According to some embodiments of the invention, the curable formulation features a viscosity at room temperature, at a shear rate of 10 1 / sec, that ranges from 250 to 720 cP. According to some embodiments of the invention, the curable formulation features a viscosity at room temperature, at a shear rate of 10 1 / sec, that ranges from 80 to 720 cP. According to a preferred embodiments described herein, the curable formulation features a viscosity at room temperature, at a shear rate of 10 1 / sec, that ranges from 80 to 250cP or from 266 to 294cP or from 952 to 963cP.

[0241] A curable formulation typically comprises one or more curable materials.

[0242] A curable material is typically hardened or cured by undergoing polymerization and / or cross -linking.

[0243] Curable materials are typically polymerizable materials, which undergo polymerization and / or cross-linking when exposed to a suitable curing condition or a suitable curing energy (a suitable energy source). Alternatively, curable materials are thermo-responsive materials, which solidify or harden upon exposure to a temperature change (e.g., heating or cooling). Optionally, curable materials are made of small particles (e.g., nanoparticles or nanoclays) which can undergo curing to form a hardened material. Further optionally, curable materials are biological materials which undergo a reaction to form a hardened or solid material upon a biological reaction (e.g., an enzymatically-catalyzed reaction).

[0244] In some of any of the embodiments described herein, a curable material is a photopolymerizable material, which polymerizes and / or undergoes cross-linking upon exposure to radiation, as described herein, and in some embodiments the curable material is a UV-curable material, which polymerizes or undergoes cross-linking upon exposure to UV-vis radiation, as described herein.

[0245] In some of any of the embodiments described herein, when a curable material is exposed to a curing condition (e.g., radiation), it polymerizes by any one, or combination, of chain elongation, entanglement and cross-linking. The cross-linking can be chemical and / or physical.

[0246] In some of any of the embodiments described herein, a curable material can be a monofunctional curable material or a multi-functional curable material.

[0247] Herein, a mono-functional curable material comprises one curable group - a functional group that can undergo polymerization, entanglement and / or cross-linking when exposed to a curing condition (e.g., radiation, presence of calcium ions).

[0248] A multi-functional curable material comprises two or more, e.g., 2, 3, 4 or more, curable groups. Multi-functional curable materials can be, for example, di-functional, tri-functional or tetra-functional curable materials, which comprise 2, 3 or 4 curable groups, respectively.

[0249] According to some of any of the embodiments described herein, the formulation comprises one or more photocurable materials, preferably UV-curable materials, which comprise one or more UV-curable groups.

[0250] In some of any of the embodiments described herein, at least a portion of the photocurable groups, are (meth)acrylic groups, such that the one or more of the photocurable materials include one or more acrylic materials.

[0251] Herein throughout, an acrylic material is used to collectively describe material featuring one or more acrylate, methacrylate, acrylamide and / or methacrylamide group(s).

[0252] Similarly, an acrylic group is used to collectively describe curable groups which are acrylate, methacrylate, acrylamide and / or methacrylamide group(s), collectively referred to herein also as (meth)acrylic groups.

[0253] Herein throughout, the term “(meth) acrylic” encompasses acrylic and methacrylic materials.

[0254] According to some of any of the embodiments described herein, the photocurable materials included in the formulation are biocompatible photocurable materials, which can be materials derived from a biological material and / or are biocompatible synthetic materials.

[0255] A formulation that comprises such photocurable materials is also referred to herein as a bioink formulation or bioink composition or simply as bioink.

[0256] According to some embodiments, the curable formulation is usable in additive manufacturing of a 3D object as described herein (e.g., in bioprinting). According to some embodiments, the composition is usable, or is for use, in the preparation of, or as, one or more modeling material formulation(s) for an additive manufacturing process (e.g., bioprinting). According to some embodiments, the additive manufacturing is of a three-dimensional object that comprises, in at least a portion thereof, a biological material, for example, a collagen material as described herein.

[0257] When the formulation comprises two or more photocurable materials, each material can feature one or more types of photocurable groups, and the photocurable groups in each material can be the same or different.

[0258] According to some of any of the embodiments described herein, the one or more biocompatible photocurable materials comprise a curable collagen.

[0259] The term "collagen" as used herein, refers to a polypeptide having a triple helix structure and containing a repeating Gly-X- Y triplet, where X and Y can be any amino acid but are frequently the amino acids proline and hydroxyproline. According to one embodiment, the collagen is a type I, II, III, V, XI, or biologically active fragments therefrom.

[0260] A collagen according to some of the present embodiments also refers to homologs (e.g., polypeptides which 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 say 100 % homologous to collagen sequences such as listed in Table A as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters). The homolog may also refer to a deletion, insertion, or substitution variant, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof.

[0261] According to some embodiment, the collagen is a human or animal-derived collagen, such as from human cadaver or animal sources (bovine, porcine, equine etc.). According to a particular embodiment, the collagen is a human collagen.

[0262] In another embodiment, the collagen comprises a naturally occurring amino acid sequence of human collagen.

[0263] Table A below lists examples of collagen NCBI sequence numbers.

[0264] Table A The annotation of SEQ ID NO: 1 is as follows:

[0265] Amino acids 1-22 - signal peptide;

[0266] Amino acids 23-161 - N-terminal peptide;

[0267] Amino acids 162-1218 - collagen alpha- 1(1) chain;

[0268] Amino acids 1219-1464 - C -terminal peptide;

[0269] The annotation of SEQ ID NO: 2 is as follows:

[0270] Amino acids 1-22 - signal peptide;

[0271] Amino acids 23-79 - N-terminal peptide;

[0272] Amino acids 80-1119 - collagen alpha-2(I) chain;

[0273] Amino acids 1120-1366 - C-terminal peptide.

[0274] According to one embodiment, the collagen comprises a sufficient portion of its telopeptides such that under suitable conditions it is capable of forming fibrils.

[0275] Thus, for example, the collagen may be atelocollagen, a telocollagen or procollagen.

[0276] As used herein, the term "atelocollagen" refers to collagen molecules lacking both the N- and C-terminal propeptides typically comprised in procollagen and at least a portion of its telopeptides, but including a sufficient portion of its telopeptides such that under suitable conditions it is capable of forming fibrils.

[0277] The term "procollagen" as used herein, refers to a collagen molecule (e.g., human) that comprises either an N-terminal propeptide, a C-terminal propeptide or both. Exemplary human procollagen amino acid sequences are set forth by SEQ ID NOs: 3, 4, 5 and 6.

[0278] The term "telocollagen" as used herein, refers to collagen molecules that lack both the N- and C-terminal propeptides typically comprised in procollagen but still contain the telopeptides. The telopeptides of fibrillar collagen are the remnants of the N-and C-terminal propeptides following digestion with native N / C proteinases.

[0279] According to another embodiment, the collagen is devoid of its telopeptides and is not capable of undergoing fibrillogenesis.

[0280] According to another embodiment, the collagen is a mixture of the types of collagen above.

[0281] According to a particular embodiment, the collagen is genetically engineered using recombinant DNA technology (e.g., human collagen).

[0282] Methods of isolating collagen from animals are known in the art. Dispersal and solubilization of native animal collagen can be achieved using various proteolytic enzymes (such as porcine mucosal pepsin, bromelain, chymopapain, chymotrypsin, collagenase, ficin, papain, peptidase, proteinase A, proteinase K, trypsin, microbial proteases, and, similar enzymes or combinations of such enzymes) which disrupt the intermolecular bonds and remove the immunogenic non-helical telopeptides without affecting the basic, rigid triple-helical structure which imparts the desired characteristics of collagen (see U.S. Pat. Nos. 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037; 3,949,073; 4,233,360 and 4,488,911 for general methods for preparing purified soluble collagen). The resulting soluble collagen can be subsequently purified by repeated precipitation at low pH and high ionic strength, followed by washing and resolubilization at low pH.

[0283] Plants expressing collagen chains and procollagen are known in the art, see for example, International Patent WO 2006 / 035442; Merle et al., FEBS Lett. 2002 Mar 27;515(l-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 Applications Publication Nos. 2002 / 098578 and 2002 / 0142391, as well as U.S. Patent No. 6,617,431.

[0284] It will be appreciated that embodiments of the present invention also contemplate genetically modified forms of collagen / atelocollagen - for example collagenase-resistant collagens and the like [see, for example, Wu et al., Proc Natl. Acad Sci, Vol. 87, p. 5888-5892, 1990],

[0285] Recombinant procollagen or telocollagen (e.g., human) may be expressed in any nonanimal cell, including but not limited to plant cells and other eukaryotic cells such as yeast and fungus.

[0286] Plants in which procollagen or telocollagen may be produced (i.e., expressed) may be of lower (e.g., moss and algae) or higher (vascular) plant species, including tissues or isolated cells and extracts thereof (e.g., cell suspensions). Preferred plants are those which are capable of accumulating large amounts of collagen chains, collagen and / or the processing enzymes described herein below. Such plants may also be selected according to their resistance to stress conditions and the ease at which expressed components or assembled collagen can be extracted. Examples of plants in which human procollagen may be expressed include, but are not limited to tobacco, maize, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, lettuce and cotton.

[0287] Production of recombinant procollagen is typically effected by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen.

[0288] In some of any of the embodiments described herein, the recombinant human collagen is a recombinant human type I collagen.

[0289] In some of any of the embodiments described herein, the recombinant human collagen is a plant-derived recombinant human collagen and in some embodiments the plant is tobacco. An exemplary collagen is 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. In some of any of the embodiments described herein, the recombinant human collagen is a recombinant human type I collagen comprising two al units having the amino acid sequence which is at least 90 % homologous, at least 91 % homologous, 92 % homologous, at least 93 % homologous, at least 94 % homologous, at least 95 % homologous, at least 96 % homologous, at least 97 % homologous, at least 98 % homologous, at least 99 % homologous or 100 % homologous to the sequence as set forth in SEQ ID NO: 15 as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters), and one a2 unit having the amino acid sequence which is at least 90 % homologous, at least 91 % homologous, 92 % homologous, at least 93 % homologous, at least 94 % homologous, at least 95 % homologous, at least 96 % homologous, at least 97 % homologous, at least 98 % homologous, at least 99 % homologous or 100 % homologous to the sequence as set forth in SEQ ID NO:6. According to a particular embodiment, the type I collagen consists of two al units which consists of the sequence as set forth in SEQ ID NO: 15 and one a2 unit consisting of the sequence as set forth in SEQ ID NO: 6, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters).

[0290] In some of any of the embodiments described herein, the al unit is encoded by a polynucleotide sequence being at least which 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 as set forth in SEQ ID NO: 16. The a2 unit is encoded by a polynucleotide sequence being at least which 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 as set forth in SEQ ID NO: 10.

[0291] Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

[0292] In some of any of the embodiments described herein, the human recombinant collagen (rhCollagen) as described herein in any of the respective embodiments is a monomeric rhCollagen. By “monomeric” it is meant a rhCollagen as described herein which is soluble in an aqueous solution and does not form fibrillar aggregates.

[0293] In some of any of the embodiments described herein, the human recombinant collagen (rhCollagen) as described herein in any of the respective embodiments is a fibrillar rhCollagen.

[0294] By “fibrillar” it is meant a rhCollagen as described herein which is in a form of fibrillar aggregates in an aqueous solution containing same. Typically, but not obligatory, fibrillar rhCollagen is formed by subjecting monomeric rhCollagen to a fibrillogenesis buffer, typically featuring a basic pH. An exemplary procedure for forming fibrillar rhCollagen, is described in WO 2018 / 225076.

[0295] As described therein plant-derived rhCollagen, is a bioidentical to human collagen and offers high purity, homogeneity, biofunctionality biocompatibility and reproducibility thereby facilitating mass production. As rhCollagen is not sourced from animals it eliminates the risks of animal-derived materials and supports a more ethical, animal-free approach to regenerative medicine.

[0296] According to some of any of the embodiments described herein, the collagen is a tissue- derived collagen, for example, a tissue-derived human Type I collagen.

[0297] By “curable collagen” it is meant a collagen as described herein in any of the respective embodiments (e.g., human recombinant collagen), which features one or more curable groups as defined herein. According to some of any of the embodiments described herein, the curable collagen is a multi-functional curable material that comprises a plurality of curable groups, as defined herein.

[0298] The terms “curable collagen” and “collagen featuring one or more (or at least one) curable groups” are used herein interchangeably.

[0299] According to some of any of the embodiments described herein, the curable collagen comprises an amino acid sequence as described herein in any of the respective embodiments, and features one or more, preferably a plurality of, curable groups generated at least a portion of the amino acid residues forming the collagen, preferably by covalent attachment of a compound that comprises a curable group to functional groups of the side chains of the amino acid residues. Alternatively, or in addition, curable groups can be generated at the N-terminus and / or C-terminus of one or more the units forming the collagen, for example, by covalent attachment of a compound that comprises a curable group to a respective amine or carboxylate.

[0300] According to some of any of the embodiments described herein, the curable collagen is as described in WO 2018 / 225076. According to some of any of the embodiments described herein, a curable collagen describes a collagen as described herein (e.g., rhCollagen as described herein in any of the respective embodiments) to which one or more curable groups are attached directly (e.g., by means of a covalent bond to a respective lysine residue of the collagen), or are not attached by means of an elastic moiety that terminates by a curable group as described herein.

[0301] According to some of any of the embodiments described herein, at least a portion of the curable groups in a curable collagen as described herein are cross -linkable photocurable groups, which undergo cross-linking when exposed to irradiation as the curing condition.

[0302] In some embodiments, curable groups can undergo polymerization and / or cross-linking via free -radical mechanism.

[0303] Exemplary curable groups include acrylic groups, including acrylate, methacrylate, acrylamide and methacrylamide groups, which are collectively referred to herein as (meth)acrylic groups. Other free-radical curable groups may include thiols, vinyl ethers and other groups that feature a reactive double bond.

[0304] In some embodiments, curable groups can undergo polymerization and / or cross-linking via other mechanisms, such as cationic polymerization, or (cationic or anionic) ring opening polymerization. Exemplary such curable groups include, but are not limited to, epoxy-containing groups, caprolactam, caprolactone, oxetane, and vinyl ether.

[0305] Other curable groups can include, for example, formation of amide bonds between functional carboxylate and amine group (each being a curable group that reacts with the other and can effect cross-linking); formation of an imine bond between and amine and an aldehyde group; formation of urethane between isocyanate groups and hydroxyl groups via polycondensation in the presence of a catalyst and / or upon exposure to UV radiation; and formation of disulfide bonds between two thiols.

[0306] Any other photocurable groups are contemplated.

[0307] The photocurable groups in the curable collagen can be generated by means of chemical reactions between a material that comprises or can generate the photocurable group(s) when reacted with chemically-compatible functional groups present in the collagen, as described herein, either directly, or be means of a spacer or a linker, using chemistries well known in the art. For example, a material that comprises a curable group and a functional group can be reacted with a compatible functional group in the collagen, for example, a functional group in an amino acid side chain, such that the curable group is a substituent of the amino acid side chain. In some embodiments, a compatible functional group is first generated within the collagen by chemical modification of chemical groups of the collagen, and is then reacted with a material that comprises or generates a curable group upon the reaction.

[0308] Whenever a curable collagen comprises more than one photocurable groups, the photocurable groups can be the same or different.

[0309] According to some of any of the embodiments described herein, at least a portion, or all, of the curable groups in a curable collagen of the present embodiments are photopolymerizable groups (e.g., UV-curable groups) that are capable of undergoing polymerization and / or crosslinking upon exposure to irradiation as described herein.

[0310] According to some of any of the embodiments described herein the curable group is a photocurable or photopolymerizable group (e.g., a (meth)acrylic group such as an acrylate or methacrylate).

[0311] Alternatively, one or more of the curable groups is a thiol-containing group, which provides disulfide bridge upon curing.

[0312] Alternatively, one or more of the curable groups is cured upon undergoing a chemical reaction, such as glycation or conjugation (using coupling agents such as EDC).

[0313] According to some embodiments, one or more of the curable groups comprise an amine and a carboxyl group which form peptide bonds upon curing.

[0314] According to some of any of the embodiments described herein, at least a portion, or all, of the curable groups in a curable collagen of the present embodiments are (meth)acrylic groups, as defined herein.

[0315] According to some of any of the embodiments described herein, an acrylic group such as methacrylamide can be generated by reacting an acrylate or methacrylate (e.g., acrylic acid, methacrylic acid, acrylic or methacrylic ester, acrylic or methacrylic anhydride) with an amine functional group (of, for example, lysine residues).

[0316] According to some of any of the embodiments of the present invention, the number of the curable groups in a curable collagen as described herein can determine the degree of curing (e.g., the degree of cross-linking) and can be manipulated in order to achieve a desired curing (e.g., crosslinking) degree.

[0317] According to some of any of the embodiments described herein, the curable collagen features a plurality of acrylamide or methacrylamide curable groups generated by reacting with lysine residues as described herein. According to some of any of the embodiments described herein, the curable collagen features a plurality of acrylamide or methacrylamide curable groups substituting the amine groups of lysine residues in the collagen.

[0318] 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 %, or at least 80 %, or at least 90 % of the lysine residues in the collagen are substituted by a methacrylamide or acrylamide group. In some embodiments, the curable collagen features from 10 % to 90 %, or from 10 % to 80 %, or from 10 % to 60 %, or from 10 to 50 %, or from 20 to 90 %, or from 20 to 80 %, or from 20 to 60 %, or from 20 to 50 %, of its lysine residues substituted by a methacrylamide or acrylamide group, including any intermediate values and subranges therebetween.

[0319] In a particular embodiment, the curable collagen is 50 % methacrylated.

[0320] In another embodiment, the curable collagen is 90 % methacrylated.

[0321] A curable collagen (e.g., rhCollagen) as described herein can be prepared by reacting a material that comprises a curable group or which generates a curable group with the collagen (e.g., rhCollagen), as described, for example, in WO 2018 / 225076. The number of curable groups in the collagen (e.g., rhCollagen) can be controlled by manipulating the amount of the material reacted with the collagen (e.g., rhCollagen) for generating the curable groups.

[0322] According to some of any of the embodiments described herein, the curable collagen is a recombinant human type I collagen as described herein in any of the respective embodiments and any combination thereof.

[0323] According to some of any of the embodiments described herein, the curable collagen is an elastomeric recombinant human type I collagen, as described, for example, in WO2023 / 073711.

[0324] Contemplated amounts of curable collagen in the formulations are between 0.1-2 % by weight of the total weight of the formulation. It will be appreciated, when the formulation lacks a polymer, the amount of curable collagen (e.g. CMR50 or CMR90) in the formulation is between 1-2 %. When the formulation comprises a polymer, the amount of curable collagen (e.g. CMR50, Colima or Gelma) may be between 0.1-1 % by weight of the total weight of the formulation.

[0325] As mentioned, the curable compositions described herein also comprise a photoinitiator.

[0326] The photoinitiator is selected in accordance with the curing mechanism (e.g., free-radical, cationic, etc.) and as suitable for the irradiation wavelength or wavelength range.

[0327] A free-radical photoinitiator may be any compound that produces a free radical on exposure to radiation such as ultraviolet or visible radiation and thereby initiates a polymerization reaction. Non-limiting examples of suitable photoinitiators include benzophenones (aromatic ketones) such as benzophenone, methyl benzophenone, Michler's ketone and xanthones; acylphosphine oxide type photo-initiators such as 2,4,6-trimethylbenzolydiphenyl phosphine oxide (TMPO) and salts thereof (e.g., Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Sodium phenyl-2,4,6- trimethylbenzoylphosphinate (NAP)) 2,4,6-trimethylbenzoylethoxyphenyl phosphine oxide (TEPO) and salts thereof, and bisacylphosphine oxides (B APO's) and salts thereof); benzoins and bezoin alkyl ethers such as benzoin, benzoin methyl ether and benzoin isopropyl ether and the like. Examples of photoinitiators are alpha-amino ketone, and bisacylphosphine oxide (B APO's).

[0328] Exemplary photoinitiators include, but are not limited to, those of the Irgacure® family, riboflavin, rose Bengal, and more.

[0329] A free-radical photo-initiator may be used alone or in combination with a co-initiator. Coinitiators are used with initiators that need a second molecule to produce a radical that is active in the photocurable free-radical systems. Benzophenone is an example of a photoinitiator that requires a second molecule, such as an amine, to produce a free radical. After absorbing radiation, benzophenone reacts with a ternary amine by hydrogen abstraction, to generate an alpha-amino radical which initiates polymerization of acrylates. Non-limiting examples of a class of coinitiators are alkanolamines such as triethylamine, methyldiethanolamine and triethanolamine.

[0330] Suitable cationic photoinitiators include, for example, compounds which form aprotic acids or Bronsted acids upon exposure to ultraviolet and / or visible light sufficient to initiate polymerization. The photoinitiator used may be a single compound, a mixture of two or more active compounds, or a combination of two or more different compounds, i.e., co-initiators. Non-limiting examples of suitable cationic photoinitiators include aryldiazonium salts, diaryliodonium salts, triarylsulphonium salts, triarylselenonium salts and the like. An exemplary cationic photoinitiator is a mixture of triarylsolfonium hexafluoroantimonate salts.

[0331] Non-limiting examples of suitable cationic photoinitiators include P-(octyloxyphenyl) phenyliodonium hexafluoroantimonate UVACURE 1600 from Cytec Company (USA), iodonium (4-methylphenyl)(4-(2-methylpropyl)phenyl)-hexafluorophosphate known as Irgacure 250 or Irgacure 270 available from Ciba Speciality Chemicals (Switzerland), mixed arylsulfonium hexafluoroantimonate salts known as UVI 6976 and 6992 available from Lambson Fine Chemicals (England), 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), iodonium bis(4-dodecylphenyl)- (OC-6-1 l)-hexafluoro antimonate known as Tego PC 1466 from Evonik Industries AG (Germany).

[0332] According to some of any of the embodiments described herein, the photoinitiator is a free- radical photoinitiator, as described herein, for example, a photoinitiator of the acylphosphine oxide type, such as, for example, Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and / or Sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP).

[0333] According to some of any of the embodiments described herein, the photoinitiator is an acyl phosphine oxide type photoinitiator such as a 2,4,6-trimethylbenzolydiphenyl phosphine oxide (TMPO) or a salt thereof (e.g., Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Sodium pheny 1-2 ,4 , 6-trimethy Ibenzoy Ipho sphinate (N AP) , 2,4 , 6-trimethy Ibenzoy lethoxypheny 1 phosphine oxide (TEPO), and bisacylphosphine oxides (B APO's).

[0334] According to some of any of the embodiments described herein, an amount of the 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, % by weight, including any intermediate values and subranges therebetween.

[0335] The amount of the photoinitiator can be determined in accordance with additive manufacturing method, the amount of photoabsorber, light intensity, the layer thickness, the desired resolution and the viscosity suitable for the AM technique.

[0336] Other curable materials that can be included in the curable formulation, optionally in combination with a curable collagen as described herein, can be any photocurable biocompatible materials.

[0337] According to some of any of the embodiments described herein, the curable formulation provides, when hardened, a hydrogel material, formed upon cross-linking the photocurable materials within an aqueous carrier such as described herein.

[0338] Herein and in the art, the term “hydrogel” describes a three-dimensional fibrous network containing at least 20 %, typically at least 50 %, or at least 80 %, and up to about 99.99 % (by mass) water. A hydrogel can be regarded as a material which is mostly water, yet behaves like a solid or semi-solid due to a three-dimensional crosslinked solid-like network, made of polymeric chains (e.g., collagen chains), within the liquid dispersing medium. The polymeric chains are inter-connected (crosslinked) by chemical bonds (covalent, hydrogen and ionic / complex / metallic bonds, typically covalent bonds).

[0339] Herein throughout, whenever polymeric chains or a polymeric material is described, it encompasses a polymeric biological materials (e.g., macromolecules) such as peptides, proteins, oligonucleotides and nucleic acids.

[0340] Hydrogels may take a physical form that ranges from soft, brittle and weak to hard, elastic and tough material. Soft hydrogels may be characterized by rheological parameters including elastic and viscoelastic parameters, while hard hydrogels are suitably characterized by tensile strength parameters, elastic, storage and loss moduli, as these terms are known in the art. The softness / hardness of a hydrogel is governed inter alia by the chemical composition of the polymer chains, the “degree of cross-linking” (number of interconnected links between the chains), the aqueous media content and composition, and temperature.

[0341] A hydrogel, according to some embodiments of the present invention, may contain macromolecular polymeric and / or fibrous elements which are not chemically connected to the main crosslinked network but are rather mechanically intertwined therewith and / or immersed therein. Such macromolecular fibrous elements can be woven (as in, for example, a mesh structure), or non-woven, and can, in some embodiments, serve as reinforcing materials of the hydrogel’s fibrous network. Non-limiting examples of such macromolecules include polycaprolactone, gelatin, crosslinked gelatin formed of, for example, gelatin methacrylate, alginate, cross-linked alginate formed of, for example, alginate methacrylate, chitosan, cross-linked chitosan formed of, for example, chitosan methacrylate, glycol chitosan, cross-linked glycol chitosan from of, for example, glycol chitosan methacrylate, hyaluronic acid (HA), cross-linked hyaluronic acid form of, for example, HA methacrylate, and other cross-linked or non-crosslinked natural or synthetic polymeric chains and the likes. Alternatively, or in addition, such macromolecules are chemically connected to the main crosslinked network of the hydrogel, for example, by acting as a cross-linking agent, or by otherwise forming a part of the three-dimensional network of the hydrogel.

[0342] 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 at the nanoscale range.

[0343] According to some of any of the embodiments described herein, the curable formulation further comprises one or more additional materials, including, for example, one or more non- curable materials and / or one or more biological components or materials.

[0344] The hydrogel, according to embodiments of the present invention, can be of biological origin or synthetically prepared.

[0345] According to some embodiments of the present invention, the hydrogel is biocompatible, and is such that when a biological moiety is impregnated or accumulated therein, an activity of the biological moiety is maintained, that is, a change in an activity of the biological moiety is no more than 30 %, or no more than 20 %, or no more than 10 %, compared to an activity of the biological moiety in a physiological medium.

[0346] Exemplary polymers or co-polymers usable for forming a hydrogel according to the present embodiments include but are not limited to polyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides, polyvinylpyrrolidone and copolymers of any of the foregoing. Other examples include polyethers, polyurethanes, and poly(ethylene glycol), functionalized by cross-linking (e.g., curable) groups or usable in combination with compatible cross linking agents.

[0347] Some specific, non-limiting examples, include: poly(2-vinylpiridine), poly(acrylic acid), poly(methacrylic acid), poly(N-isopropylacrylamide), poly(N,N’-methylenbisacrylamide), poly(N-(N-propyl)acrylamide), poly(methacyclic acid), poly (2-hydroxy acrylamide), poly(ethylene glycol) acrylate, poly(ethylene glycol) methacrylate, and polysaccharides such as hyaluronic acid, dextran, alginate, agarose, and the like, and any co-polymer of the foregoing.

[0348] Hydrogel precursors (hydrogel-forming materials) forming such polymeric chains are contemplated, including any combination thereof.

[0349] Hydrogels are typically formed of, or are formed in the presence of, di- or tri- or multifunctional monomers, oligomer or polymers, which are collectively referred to as hydrogel precursors or hydrogel-forming agents or hydrogen-forming materials, having two, three or more polymerizable groups. The presence of more than one polymerizable group renders such precursors cross-linkable, and allow the formation of the three-dimensional network.

[0350] Exemplary cross -linkable monomers include, without limitation, the family of di- and triacrylates monomers, which have two or three polymerizable functionalities, one of which can be regarded as a cross-linkable functional group. Exemplary diacrylates monomers include, without limitation, methylene diacrylate, and the family of poly(ethylene glycol)ndimethacrylate (nEGDMA). Exemplary triacrylates monomers include, without limitation, trimethylolpropane triacrylate, pentaerythritol triacrylate, tris (2-hydroxy ethyl) isocyanurate triacrylate, isocyanuric acid tris(2-acryloyloxyethyl) ester, ethoxylated trimethylolpropane triacrylate, pentaerythrityl triacrylate and glycerol triacrylate, phosphinylidynetris(oxyethylene) triacrylate.

[0351] In some of any of the embodiments described herein, a curable material, whether monomeric or oligomeric, can be a mono-functional curable material or a multi-functional curable material.

[0352] Curable materials usable in the field of bioprinting are predominantly based on either naturally derived materials, including, for example, Matrigel, Alginate, Pectin, Xanthan gum, Gelatin, Chitosan, Fibrin, Cellulose and Hyaluronic acid, which can be isolated from animal or human tissues, or recombinantly-generated, or synthetically-prepared materials, including, for example, poly (ethyleneglycol), PEG, gelatin methacrylate; GelMA, polypropylene oxide); PPG, poly(ethylene oxide); PEG; poly(ethyleneglycol)methacrylate (PEG-MA), polyethyleneglycoldiacrylate (PEG-DA), poly(ethyleneglycol)dimethylacrylate (PEGDMA), polyglutamic acid, PLGA / PLLA, poly(dimethyl siloxane); Nanocellulose; Pluronic F127, short di-peptides (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).

[0353] Exemplary curable materials usable in the context of the present embodiments include, but are not limited to, Matrigel, Gelatin methacrylate (GelMA), Nanocellulose (nano-scaled structured materials which are UV-curable, including cellulose nanocrystals (CNC), cellulose nanofibrils (CNF), and bacterial cellulose (BC), also referred to as microbial cellulose), Pluronic® materials, including, for example, Pluronic F127 which is fluid at a low temperature forms a gel at a high temperature, above critical micellar concentration (CMC) and Pluronic Fl 27 -diacrylate (DA) which is UV-curable, Hyaluronic acid (HA), Acrylated hyaluronic acid (AHA), methacrylated hyaluronic acid (MAHA), Poly-(ethylene glycol) diacrylate (PEGDA),poly(ethyleneglycol)methacrylate (PEG-MA), poly(ethyleneglycol)dimethylacrylate (PEGDMA), Alginate, Xanthan gum, Pectin, Chitosan which can be crosslinked with a chemical agent such as Glutaraldehyde, Genipin or Sodium Tripolyphosphate (TPP).

[0354] According to some of any of the embodiments described herein, the curable material is or comprises a poly(alkylene glycol) such as a polyethylene glycol) that features one or more photocurable group(s), for example, one or more acrylic group(s) as described herein. In some of these embodiments, the curable material can be, for example, a poly(alkylene glycol) (meth)acrylate such as a poly(ethylene glycol) (meth)acrylate, and / or a poly(alkylene glycol) di(meth)acrylate such as a poly(ethylene glycol) di(meth)acrylate, and / or a copolymer that comprises the foregoing, for example, poly caprolactone (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 foregoing.

[0355] According to some of any of the embodiments described herein, the curable material is or comprises a poly(alkylene glycol) that features at least one (meth)acrylic group at its terminus, that is, it is a polymer or copolymer of poly(alkylene glycol) that features at least one (meth)acrylic group at its terminus.

[0356] According to some of any of the embodiments described herein, a poly(ethylene glycol) that features curable group(s) or a co-polymer thereof has an average molecular weight of at least 500 grams / mol or at least 700 grams / mol. In some embodiments, the average molecular weight is lower than 4,000, or lower than 3,000 or lower than 2,000, or lower than 1,000 grams / mol. In some embodiments, the average molecular weight ranges from 500 to 40,000, or 500 to 30,000, or from 500 to 20,000, or from 50 to 10,000, or from 500 to 5,000, or from 500 to 4,000, or from 500 to 3,000, or from 500 to 2,000, grams / mol, including any intermediate values and subranges therebetween. In some embodiments, the average molecular weight ranges from 3,000 to about 40,000, or 3,000 to about 30,000, or from 3,000 to about 20,000, or from 3,000 to about 10,000, grams / mol, including any intermediate values and subranges therebetween.

[0357] According to some of any of the embodiments described herein, a concentration of each of the curable materials in the formulation ranges from 1 to 30, or from 1 to 20, or from 1 to 10, or from 1 to 8, or from 1 to 5, or from 2 to 4, or from 7 to 9, or from 14 to 16, or from 2.5 to 7.5, or from 5 to 20, or from 5 to 15, or from 10 to 20, or from 10 to 30, % by weight of the total weight of the composition, including any intermediate values and subranges therebetween.

[0358] In some of any of the embodiments described herein, a concentration of each of the curable materials as described herein in any of the respective embodiments and any combination thereof in the curable formulation ranges from 0.5 mg / mL to 50 mg / mL, or from 0.5 mg / mL to 20 mg / mL, or from 1 mg / mL to 50 mg / mL, or from 1 mg / mL to 40 mg / mL, or from 1 mg / mL to 30 mg / mL, or from 1 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, including any intermediate values and subranges therebetween.

[0359] A concentration of the curable materials in a curable formulation containing the same can affect the rheological properties of the formulation and of the hardened material obtained upon curing (upon exposure to a curing condition such as, for example, irradiation), and can be manipulated in accordance with the AM methodology and conditions employed and desired properties of the final object or a portion thereof.

[0360] According to the present embodiments, the formulation further comprises a mixture of photocurable polymeric materials that, when combined with the photocurable biological or biocompatible material, and exposed to irradiation as a curing condition, provides a hardened material that features mechanical properties suitable for or meeting the requirements of a breast implant.

[0361] According to some of any of the embodiments described herein, the curable formulation provides, when hardened (e.g., when used in an additive manufacturing process as described herein and following photocuring and terminal sterilization), a biological or a biocompatible implant featuring one or more of: a. stiffness characterized by volume retention under physiological stress of no more than 30 % strain, at 10 N force, as determined in a compression force versus strain measurements as described herein; b. compressive strain-at-break higher than 50% (as determined in a compression force versus strain measurements as described herein); and c. compressive force limit of at least 156 N (as determined in a compression force versus strain measurements as described herein). According to some of any of the embodiments described herein, the curable formulation provides, when hardened or cured (e.g., when used in an additive manufacturing process as described herein and following photocuring and washing (“as print”) and optionally post process, a hardened or cured material which exhibits tensile strain-at-break of at least 70 %; and tensile stress of at least 0.7 MPa or even at least IMPa (as determined in stress versus strain measurements as described herein (e.g., for dog bone). For example, at 70 % strain, the material exhibits tensile stress of 0.7 MPa, or of 0.75 MPa, or of 0.8 MPa, or higher. For example, at 80 % strain, the material exhibits stress of 0.7 MPa, or of 0.75 MPa, or of 0.8 MPa, or higher. For example, at 100 % strain, the material exhibits tensile stress of 0.7 MPa, or of 0.75 MPa, or of 0.8 MPa, or of 0.85 MPa, or of 0.9 MPa, or of 0.95 MPa, or of 1.00 MPa, or higher. For example, at 110 % strain, the material exhibits tensile stress of 0.7 MPa, or of 0.75 MPa, or of 0.8 MPa, or of 0.85 MPa, or of 0.9 MPa, or of 0.95 MPa, or of 1.00 MPa, or of 1.10 MPa, or of 1.15 MPa, or of 1.20 MPa, or higher. All when measured as described herein.

[0362] Stiffness is represented by a force-strain curve measured in a compression force test as described herein, such that strain of 50 % at 100-200 N, means the force required to compress the implant by 50% should be in the range of 100-200 N; and strain of 20 % at 10-100 N, means the force required to compress the implant by 20% should be in the range of 10-100 N.

[0363] The stiffness of a specimen (or lattice) is represented by the slope of the measured stressstrain (or load-strain) response curve. For a nonlinear curve, the stiffness is calculated using a linear regression within a given range, for example, 0-10% of strain, or 5-15% of strain, or 10- 20% of strain, or 10-30% of strain, or 10-40% of strain, or 20-50% of strain, or 40-60% of strain, or 60-70% of strain. Stiffness could also be referred to as instantaneous stiffness, especially if measured in a very narrow range such as 0-5% of strain, 5-10% of strain, and 10-15% of strain, which is common for nonlinear materials (or structures).

[0364] Compressive strain-at-break represents the strain level in which the specimen (or lattice) is partially or completely broken during a compressive test. One way to determine the breaking point of the implant is by analyzing the measured stress-strain (or force-strain) curve and determining the point where the stress (or force) starts to sharply decrease while the strain is still rising, thus being indicative of unstable mechanical response which implies on implant failure (break). Failure as such indicates an irreversible defect in the shape or integrity of the specimen (or lattice). Other smoothly, controlled fluctuations in the stress-strain (or force-strain) curves that appear during test are typically for the cylinder specimens (and breast scaffold) indicating meaningful elastic deformation named buckling. Tensile strain-at-break has the same meaning and is measured via a tensile dogbone test. Compressive strength (or force limit) is the corresponding stress (or force) value to its strain-at-break one. This value is typically the maximal stress (or force) measured in a compression material test on cylinders (or scaffold test on breast implants), but can also be lower than the maximal value as it is determined according to the moment of breakage / other failure mechanism. Tensile strength has the same meaning and measured in a tensile dogbone test.

[0365] Volume retention of a specimen (or scaffold) is its ability to withstand loads within its elastic deformation range. Meaning that once loads are removed from the specimen (or scaffold), it returns to its undeformed shape. This ability is measured in-vitro during non-destructive tests, and in-vivo while the implants are under physiological loads.

[0366] In-vitro and ex-vivo, load and displacement are continuously accurately measured using mechanical and electronical components of a universal tester machine, Instron 68SC-1, capable of conducting tests with loads up to 1000N. Test methods were defined in Instron’ s dedicated software, Bluehill, controlling test parameters such as cross-head displacement speed, preload cycling properties, and end-of-test conditions. Standards such as ASTM F2150-19, ASTM D695, and ASTM D638 can be used to guide the test protocol and methods.

[0367] An exemplary apparatus usable for the compression and stress / strain measurements is described in WO 2021 / 191897 (see FIG. 1A-B therein).

[0368] According to the embodiments described herein, a curable polymeric material is a photopolymerizable or photocurable material, which polymerizes and / or undergoes cross-linking upon exposure to radiation, as described herein, and in some embodiments the curable material is a UV-curable material, which polymerizes or undergoes cross-linking upon exposure to UV-vis radiation, as described herein.

[0369] According to some of any of the embodiments described herein, the curable polymeric materials are photocurable polymeric materials, preferably UV-curable polymeric materials, which comprise one or more UV-curable groups.

[0370] It is to be understood that the photocurable polymeric materials according to these embodiments do not encompass the curable biological material as described herein in any of the respective embodiments.

[0371] According to the present embodiments, the photocurable polymeric materials are non- proteinaceous materials.

[0372] Herein, the phrase “polymeric material” encompasses homopolymers, copolymers, and a mixture of two or more of homopolymers, two or more co-polymers or of one or more homopolymer and one or more copolymer. When all the repeating units that form the polymeric material are the same, a polymeric material is considered a homopolymeric material. When the polymeric material comprises two or more types of repeating units, which can differ from one another by the type of the backbone units that form the polymeric backbone and / or by the type of the pendant groups, the polymeric material is a co-polymeric material. Co-polymeric materials comprise two or more different types of monomeric unit which may be distributed randomly or non-randomly throughout the polymeric backbone. When different types of monomeric units are distributed non-randomly, the copolymer may be characterized by any non-random distribution, and can be, for example, an alternating copolymer, a periodic copolymer, and / or a block copolymer.

[0373] As used herein, the phrase “alternating copolymer” describes a copolymer in which two adjacent backbone units alternate in a regular sequence along the polymer chain, such that the polymer has a repeating pattern of the two distinct backbone units. Typically, in an alternating copolymer, each two adjacent backbone units are different from one another (e.g., Y1-Y2-Y1-Y2 or Y1-Y2-Y3-Y1-Y2-Y3). As used herein, the phrase “periodic copolymer” describes a copolymer in which the arrangement of monomeric units follows a repeating sequence with a defined periodicity, which may involve more than two types of monomers, creating a regular and predictable sequence along the polymer chain (e.g., Y 1-Y2-Y2-Y 1-Y2-Y2-Y 1-Y2-Y2-). As used herein, the phrase “block copolymer” describes a copolymer in which distinct blocks of repeating backbone units are linked together in segments, such that the polymer consists of large sequences ("blocks") composed of a plurality (e.g., 3 or more) of one type of backbone unit followed by a block of a plurality of another type of backbone unit (e.g., Y1-Y1-Y1-Y2-Y2-Y2).

[0374] According to a particular embodiment, the formulation comprises a mixture of a photocurable polymeric material that has an average molecular weight (Mw) lower than 10,000 grams / mol (also referred to herein as a low MW photocurable polymeric material; as low MW polymer or simply as short polymer), and another photocurable polymeric material has an average molecular weight (Mw) higher than 10,000, or higher than 15,000, grams / mol (also referred to herein as a high MW photocurable polymeric material, or high MW polymer or simply as long polymer). This formulation may be particularly advantageous for use in a breast implant since it imparts a desired softness, yet still presenting with high strength.

[0375] In another embodiment, a total amount of these photocurable polymeric materials is in a range of from 1 to 20, or from 5 to 20, or from 5 to 15, or from 7.5 to 15, from 7.5 to 12, or from 5 to 10, from 5 to 12, or from 7.5 to 10, % by weight, of the total weight of the curable formulation; and / or when a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material ranges from 1: 1 to 1:9, or from 1: 1 to 1:5. According to some of any of the embodiments described herein, a total amount (concentration) of the photocurable polymeric materials in the formulation is lower than 20, or lower than 18, or lower than 16, or lower than 14, or lower than 12 %, or lower than 10 % by weight of the total weight of the formulation.

[0376] According to some of any of the embodiments described herein, a total amount (concentration) of the photocurable polymeric materials in the formulation is at least 5, preferably at least 7, or at least 10, % by weight of the total weight of the formulation.

[0377] According to some of any of the embodiments described herein, a total amount (concentration) of the photocurable polymeric materials in the formulation ranges from 5 to 15, or from 5 to 12, or from 5 to 10, or from 7.5 to 15, or preferably from 7.5 to 12, or from 7.5 to 10, % by weight, of the total weight of the curable formulation, including any intermediate values and subranges therebetween.

[0378] According to some of any of the embodiments described herein, a total amount (concentration) of the low MW polymeric material(s) in the formulation ranges from 1 to 10, preferably from 1 to 8, or from 1 to 5, or from 1 to 3 % by weight, of the total weight of the curable formulation, including any intermediate values and subranges therebetween.

[0379] According to some of any of the embodiments described herein, a total amount (concentration) of the high MW polymeric material(s) in the formulation ranges from 5 to 12, preferably from 5 to 10, or from 5 to 8% by weight, of the total weight of the curable formulation, including any intermediate values and subranges therebetween.

[0380] According to some of any of the embodiments described herein, an amount (concentration) of the high MW polymeric material is at least the same and preferably higher than an amount (concentration) of the low MW polymeric material in the formulation.

[0381] According to some of any of the embodiments described herein, a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material ranges from 1: 1 to 1:9, or from 1: 1 to 1:5, or from 1: 1 to 1:3, or from 2:5 to 2:7, or from 1: 1 to 1:2, including any intermediate values and subranges therebetween, and can be, for example, 1:2, 2:5, 1:3, 2:7, 1:4, 2:9, or 1:5.

[0382] According to some of any of the embodiments described herein, a total amount of the high MW and low MW photocurable polymeric materials is in a range of from 5 to 15, or from 7.5 to 15, or from 5 to 10, or from 7.5 to 10, % by weight, of the total weight of the curable formulation; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material ranges from 1: 1 to 1:5, including any intermediate values and subranges therebetween. According to some of any of the embodiments described herein, a total amount of the high MW and low MW photocurable polymeric materials is in a range of from 7.5 to 15, or from 7.5 to 10, % by weight, of the total weight of the curable formulation; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material ranges from 1: 1 to 1:5, including any intermediate values and subranges therebetween.

[0383] According to some of any of the embodiments described herein, a total amount of the high MW and low MW photocurable polymeric materials is in a range of from 7.5 to 15, or from 7.5 to 10, % by weight, of the total weight of the curable formulation; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material ranges from 1:2 to 1:5, or from 1: 1 to 1:4, including any intermediate values and subranges therebetween.

[0384] According to some of any of the embodiments described herein, a total amount of the high MW and low MW photocurable polymeric materials is in a range of from 5 to 10, or from 7.5 to 10, % by weight, of the total weight of the curable formulation; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material ranges from 1: 1 to 1:5, including any intermediate values and subranges therebetween.

[0385] According to some of any of the embodiments described herein, a total amount of the high MW and low MW photocurable polymeric materials is in a range of from 5 to 10, or from 7.5 to 10, % by weight, of the total weight of the curable formulation; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material ranges from 1:2 to 1:5, or from 1: 1 to 1:4, including any intermediate values and subranges therebetween.

[0386] According to some of any of the embodiments described herein, a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material is about 1:3.

[0387] According to some of any of the embodiments described herein, a total amount of the high MW and low MW photocurable polymeric materials is in a range of from 1 to 20, or from 5 to 20, or from 5 to 15, or from 7.5 to 15, % by weight, of the total weight of the curable formulation, including any intermediate values and subranges therebetween; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material is about 1:3.

[0388] According to some of any of the embodiments described herein, a total amount of the high MW and low MW photocurable polymeric materials is in a range of from 5 to 15, or from 5 to 10, or from 7.5 to 10, or from 7.5 to 15, % by weight, of the total weight of the curable formulation, including any intermediate values and subranges therebetween; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material is about 1:3.

[0389] According to some of any of the embodiments described herein, a total amount of the high MW and low MW photocurable polymeric materials is in a range of from 5 to 10, or from 7.5 to 10, % by weight, of the total weight of the curable formulation, including any intermediate values and subranges therebetween; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material is about 1:3.

[0390] According to some of any of the embodiments described herein, a total amount of the high MW and low MW photocurable polymeric materials is in a range of from 7.5 to 10, or from 7.5 to 15, % by weight, of the total weight of the curable formulation, including any intermediate values and subranges therebetween; and a weight ratio between the low MW photocurable polymeric material and the high MW photocurable polymeric material is about 1:3.

[0391] According to some of any of the embodiments described herein, each of the photocurable polymeric materials (low and high MW polymers) is a multifunctional photocurable polymeric material that features two or more photocurable groups as described herein, e.g., (meth)acrylic groups such as (meth) acrylate groups, as described herein.

[0392] According to some of any of the embodiments described herein, each of the photocurable polymeric materials (low and high MW polymers) is independently a di-functional or a trifunctional photocurable polymeric material that features two or three photocurable groups as described herein, e.g., (meth)acrylic groups such as (meth)acrylate groups, as described herein.

[0393] According to some of any of the embodiments described herein, each of the photocurable polymeric materials (low and high MW polymers) is a di-functional photocurable polymeric material that features two photocurable groups as described herein, e.g., (meth)acrylic groups such as (meth)acrylate groups, as described herein.

[0394] The two or more photocurable groups can be terminal groups, which substitute the termini of the polymeric chain of the polymeric material, and / or can be pendant groups, substituting one or more of the backbone units of the polymeric material.

[0395] According to some of any of the embodiments described herein, the two or more photocurable groups are each a terminal group.

[0396] According to some of any of the embodiments described herein, each of the photocurable polymeric materials (low and high MW polymers) is a di-functional photocurable polymeric material that features two photocurable groups as described herein, e.g., (meth)acrylic groups such as (meth)acrylate groups, as described herein, at each terminus of the polymeric chain (in case the polymeric material is made of a linear polymer).

[0397] According to some of any of the embodiments described herein, each of the photocurable polymeric materials (low and high MW polymers) is a di-functional photocurable polymeric material that features a linear polymeric chain and two photocurable groups as described herein, e.g., (meth)acrylic groups such as (meth)acrylate groups, as described herein, at each terminus of the linear (non-branched) polymeric chain.

[0398] According to some of any of the embodiments described herein, each of the photocurable polymeric materials (low and high MW polymers) is a di-functional photocurable polymeric material that features a non-linear polymeric chain and two photocurable groups as described herein, e.g., (meth)acrylic groups such as (meth) acrylate groups, as described herein, at each terminus of the non-linear (branched) polymeric chain, and / or polymeric backbone.

[0399] According to some of any of the embodiments described herein, in each of the polymeric materials, each of the photocurable groups is independently an acrylate or methacrylate group.

[0400] For example, a di-functional high MW polymeric material can comprise two acrylate groups, or two methacrylate groups, or one acrylate group and one methacrylate group.

[0401] For example, a di-functional low MW polymeric material can comprise two acrylate groups, or two methacrylate groups, or one acrylate group and one methacrylate group.

[0402] For example, a di-functional high MW polymeric material can comprise two acrylate groups, or two methacrylate groups, and a di-functional low MW polymeric material can comprise two acrylate groups, or two methacrylate groups.

[0403] For example, a multi-functional high MW polymeric material can comprise a plurality of acrylate groups, and / or a plurality of methacrylate groups, and a multi-functional low MW polymeric material can comprise a plurality of acrylate groups, and / or a plurality of methacrylate groups.

[0404] For example, a multi-functional high MW polymeric material can comprise one or more acrylate group and plurality methacrylate groups or a plurality acrylate groups and one or more methacrylate groups.

[0405] For example, a multi-functional low MW polymeric material can comprise one or more acrylate group and plurality methacrylate groups or a plurality acrylate groups and one or more methacrylate groups.

[0406] For example, a multi-functional high MW polymeric material can comprise one or more acrylate group and plurality methacrylate groups or a plurality acrylate groups and one or more methacrylate groups, and a multi-functional low MW polymeric material can comprise one or more acrylate group and plurality methacrylate groups or a plurality acrylate groups and one or more methacrylate groups.

[0407] According to some of any of the embodiments described herein, at least one of the photocurable polymeric materials features a plurality of acrylate groups.

[0408] According to some of any of the embodiments described herein, at least one of the photocurable polymeric materials is a di-functional polymeric material that features a plurality of acrylate groups.

[0409] For example, a multi-functional high MW polymeric material can comprise a plurality of acrylate groups, or a plurality of methacrylate groups, and a multi-functional low MW polymeric material can comprise a plurality of acrylate groups.

[0410] For example, a multi-functional high MW polymeric material can comprise a plurality of acrylate groups, and a multi-functional low MW polymeric material can comprise a plurality of acrylate groups, or a plurality of methacrylate groups.

[0411] For example, a multi-functional high MW polymeric material can comprise a plurality of acrylate groups, and a multi-functional low MW polymeric material can comprise a plurality of acrylate groups.

[0412] For example, a multi-functional high MW polymeric material can comprise a plurality of methacrylate groups, and a multi-functional low MW polymeric material can comprise a plurality of acrylate groups or methacrylate groups.

[0413] For example, a multi-functional high MW polymeric material can comprise a plurality of methacrylate groups, and a multi-functional low MW polymeric material can comprise a plurality of acrylate groups.

[0414] For example, a di-functional high MW polymeric material can comprise two acrylate (e.g., terminal) groups, or two (e.g., terminal) methacrylate groups, and a di-functional low MW polymeric material can comprise two (e.g., terminal) acrylate groups.

[0415] For example, a di-functional high MW polymeric material can comprise two (e.g., terminal) acrylate groups, and a di-functional low MW polymeric material can comprise two (e.g., terminal) acrylate groups, or two (e.g., terminal) methacrylate groups.

[0416] For example, a di-functional high MW polymeric material can comprise two (e.g., terminal) acrylate groups, and a di-functional low MW polymeric material can comprise two (e.g., terminal) acrylate groups.

[0417] According to some of any of the embodiments described herein, the low MW polymeric material features a plurality of acrylate groups. According to some of any of the embodiments described herein, the low MW polymeric material is a di-functional polymeric material that features two (e.g., terminal) acrylate groups.

[0418] According to some of any of the embodiments described herein, the high MW polymeric material is a di-functional polymeric material that features two terminal acrylate or methacrylate groups and the low MW polymeric material is a di-functional polymeric material that features two terminal acrylate groups.

[0419] According to some of any of the embodiments described herein, the high MW polymeric material is a di-functional polymeric material that features two terminal methacrylate groups and the low MW polymeric material is a di-functional polymeric material that features two terminal acrylate groups.

[0420] According to some of any of the embodiments described herein, the high MW polymeric material is a di-functional polymeric material that features two terminal acrylate groups and the low MW polymeric material is a di-functional polymeric material that features two terminal acrylate groups.

[0421] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has an average molecular weight (Mw) lower than 10,000 grams / mol, or lower than 8,000, or lower than 7,000, or lower then 6,000, or lower than 5,000, or lower than 4,000 grams / mol. According to some of any of the embodiments described herein, the low MW photocurable polymeric material has an average molecular weight (Mw) lower than 3,000 grams / mol, or lower than 2,000, grams / mol.

[0422] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has an average molecular weight (Mw) in a range of from 500 to 10,000, or from 500 to 8,000, or from 500 to 6,000, or from 500 to 5,000, or from 1,000 to 10,000, or from 2,000 to 10,000, or from 1,000 to 8,000, or from 2,000 to 8,000, or from 1,000 to 6,000, or from 2,000 to 6,000, or from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 6,000 or from 3,000 to 5,000 or from 3,000 to 4,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, or from 500 to 2,000 or from 500 to 1,500, grams / mol, including any intermediate values and subranges therebetween.

[0423] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has an average molecular weight (Mw) in a range of from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 5,000 or from 3,000 to 4,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, or from 500 to 2,000 or from 500 to 1,500, grams / mol, including any intermediate values and subranges therebetween. According to some of any of the embodiments described herein, the low MW photocurable polymeric material has an average molecular weight (Mw) in a range of from 500 to 4,000, or from 500 to 3,000, or from 500 to 2,000, or from 500 to 1,500, grams / mol, including any intermediate values and subranges therebetween.

[0424] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has an average molecular weight (Mw) in range of from 500 to 1,500, or from 800 to 1,200, or from 900 to 1,100, grams / mol, including any intermediate values and subranges therebetween or of about 1,000, grams / mol.

[0425] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has an average molecular weight (Mw) in range of from 1,000 to 4,000, or from 2,000 to 4,000, or from 3,000 to 4,000, or from 3,000 to 3,500, grams / mol, including any intermediate values and subranges therebetween or of about 3,400, grams / mol.

[0426] According to some of any of the embodiments described herein, the high MW photocurable polymeric material has an average molecular weight (Mw) higher than 10,000, or higher than 15,000, or higher than 20,000, grams / mol.

[0427] According to some of any of the embodiments described herein, the high MW photocurable polymeric material has an average molecular weight (Mw) of 20,000 grams / mol or higher than 20,000, grams / mol.

[0428] According to some of any of the embodiments described herein, the high MW photocurable polymeric material has an average molecular weight (Mw) in a range of from 10,000 to 50,000, or from 10,000 to 40,000, or from 10,000 to 30,000, or from 10,000 to 25,000, or from 10,000 to 24, 000, or from 10,000 to 23,000, or from 10,000 to 22,000, 10,000 to 21,000, or from 10,000 to 20, 000, or from 25,000 to 50,000, or from 15,000 to 40,000, or from 15,000 to 30,000, or from 15,000 to 25,000, or from 20,000 to 50,000, or from 20,000 to 40,000, or from 20,000 to 30,000 or from 20,000 to 25,000, or from 20,000 to 24, 000, or from 20,000 to 23,000, or from 20,000 to 22,000, 20,000 to 21,000, grams / mol, including any intermediate values and subranges therebetween,.

[0429] According to some of any of the embodiments described herein, the high MW photocurable polymeric material has an average molecular weight (Mw) in a range of from 20,000 to 50,000, or from 20,000 to 40,000, or from 20,000 to 30,000 or from 20,000 to 25,000, grams / mol, including any intermediate values and subranges therebetween, or of about 23,000, grams / mol .

[0430] According to some of any of the embodiments described herein, the high MW photocurable polymeric material has an average molecular weight (Mw) in a range of from 15,000 to 30,000, or from 20,000 to 30,000 or from 15,000 to 25,000, or from 20,000 to 25,000, grams / mol, including any intermediate values and subranges therebetween , or of about 20,000, grams / mol.

[0431] According to some of any of the embodiments described herein, the high MW photocurable polymeric material has an average molecular weight (Mw) of about 20,000, or of about 21,000, or of about 22,000, or of about 23,000, or of about 24,000, grams / mol.

[0432] According to some of any of the embodiments described herein, each of the high and low MW polymeric materials is a biocompatible polymeric material.

[0433] According to some of any of the embodiments described herein, each of the high and low MW polymeric materials is a biodegradable polymeric material.

[0434] According to some of any of the embodiments described herein, each of the high and low MW polymeric materials is a biocompatible and biodegradable polymeric material.

[0435] According to some of any of the embodiments described herein, each of the high and low MW polymeric materials is a water-soluble or water-miscible polymeric material, and / or is included in an amount that when mixed with water in equal volumes or weights, a homogeneous solution is formed.

[0436] Herein throughout, the term “water-miscible” describes a material which is at least partially dissolvable or dispersible in water, that is, at least 50 % of the molecules move into the water upon mixture. This term encompasses the terms “water-soluble” and “water dispersible”.

[0437] Herein throughout, the term “water-soluble” describes a material that when mixed with water in equal volumes or weights, a homogeneous solution is formed.

[0438] According to some of any of the embodiments described herein, one or both of the low and high curable polymeric materials is or comprises a poly(alkylene glycol) backbone such as a poly(ethylene glycol), that features one or more photocurable group(s), for example, one or more acrylic group(s) as described herein. In some of these embodiments, the curable material can be, for example, a poly(alkylene glycol) (meth) acrylate such as a poly(ethylene glycol) (meth)acrylate, and / or a poly(alkylene glycol) di(meth)acrylate such as a poly(ethylene glycol) di(meth)acrylate, and / or a copolymer that comprises any of the foregoing, for example, a copolymer of poly caprolactone and poly (ethylene glycol), (meth)acrylate and / or di(meth)acrylate; a copolymer of poly(lactic acid) and poly(ethylene glycol) (meth)acrylate and / or di(meth)acrylate; a copolymer of poly(lactic acid co-glycolic acid) and poly(ethylene glycol) (meth)acrylate and / or di(meth)acrylate, including any combination of the foregoing.

[0439] According to some of any of the embodiments described herein, the high curable polymeric material is PEG-PCL 23K(DMA) (e.g., a dimethacrylate triblock copolymer comprising two block of PCL each featuring average MW of about 1,500 grams / mol, each attached to a terminus of a PEG polymeric chain featuring an average MW of about 20,000 grams / mol).

[0440] As used herein, the term “alkylene glycol” describes a -O-[(CR’R”)Z-O]y- group, with R’ and R” being each independently hydrogen or alkyl, and with z being an integer of from 1 to 10, preferably, from 2 to 6, more preferably 2 or 3, and y being an integer of 1 or more. Preferably R’ and R” are both hydrogen. When z is 2 and y is 1, this group is ethylene glycol. When z is 3 and y is 1, this group is propylene glycol. When y is 2-4, the alkylene glycol is referred to herein as oligo(alkylene glycol). When y is higher than 4, it is a poly(alkylene glycol) backbone. When z is 2, R and R” are each hydrogen, and y is higher than 4, and R and it is a poly(ethylene glycol) backbone.

[0441] When a poly(alkylene) glycol such as a poly(ethylene glycol) terminates by photocurable groups such as (meth) acrylate groups, the (meth)acrylate groups are attached to each terminus of the polymeric backbone.

[0442] According to some of any of the embodiments described herein, one or both of the low and high curable polymeric materials is or comprises a poly(alkylene glycol) that features at least one (meth)acrylic group at its terminus, that is, it is a polymer or copolymer of poly(alkylene glycol) that features at least one (meth)acrylic group at its terminus.

[0443] According to some of any of the embodiments described herein, one or both of the low and high curable polymeric materials is or comprises a poly(alkylene glycol) that features two (or more) (meth)acrylic group at its terminus, that is, it is a polymer or copolymer of poly(alkylene glycol) that features two (meth)acrylic group at both of its termini.

[0444] According to some of any of the embodiments described herein, each of the low and high MW polymeric materials comprise a poly(alkylene glycol) polymeric backbone.

[0445] According to some of any of the embodiments described herein, each of the low and high MW polymeric materials comprise a poly(alkylene glycol) polymeric backbone and terminates by two (meth) acrylate groups, as described herein in any of the respective embodiments and any combination thereof.

[0446] According to some of any of the embodiments described herein, the low MW photocurable polymeric material is a poly(alkylene glycol) that terminates by two (or more) acrylate or methacrylate groups.

[0447] According to some of any of the embodiments described herein, the low MW photocurable polymeric material is a poly(alkylene glycol) that terminates by two (or more) acrylate groups. According to some of any of the embodiments described herein, the low MW photocurable polymeric material is a poly(ethylene glycol) that terminates by two (or more) methacrylate groups.

[0448] According to some of any of the embodiments described herein, the low MW photocurable polymeric material is a poly(ethylene glycol) that terminates by two (or more) acrylate groups, that is, it has a polyethylene glycol) backbone as described herein, that terminates by two (or more) acrylate groups, preferably two acrylate groups.

[0449] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has a polyethylene glycol) backbone featuring an average molecular weight (Mw) lower than 10,000 grams / mol, or lower than 8,000, or lower than 7,000, or lower then 6,000, or lower than 5,000, or lower than 4,000 grams / mol. According to some of any of the embodiments described herein, the low MW photocurable polymeric material has a poly(ethylene glycol) backbone featuring an average molecular weight (Mw) lower than 3,000 grams / mol, or lower than 2,000, grams / mol.

[0450] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has a poly(ethylene glycol) backbone featuring an average molecular weight (Mw) in a range of from 500 to 10,000, or from 500 to 8,000, or from 500 to 6,000, or from 500 to 5,000, or from 1,000 to 10,000, or from 2,000 to 10,000, or from 1,000 to 8,000, or from 2,000 to 8,000, or from 1,000 to 6,000, or from 2,000 to 6,000, or from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 6,000 or from 3,000 to 5,000 or from 3,000 to 4,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, or from 500 to 2,000 or from 500 to 1,500, grams / mol, including any intermediate values and subranges therebetween.

[0451] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has a poly(ethylene glycol) backbone featuring an average molecular weight (Mw) in a range of from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 5,000 or from 3,000 to 4,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, or from 500 to 2,000 or from 500 to 1,500, grams / mol, including any intermediate values and subranges therebetween.

[0452] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has a poly(ethylene glycol) backbone featuring an average molecular weight (Mw) in a range of from 500 to 4,000, or from 500 to 3,000, or from 500 to 2,000, or from 500 to 1,500, grams / mol, including any intermediate values and subranges therebetween.

[0453] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has a poly(ethylene glycol) backbone featuring an average molecular weight (Mw) in range of from 500 to 1,500, or from 800 to 1,200, or from 900 to 1,100, grams / mol, including any intermediate values and subranges therebetween or of about 1,000, grams / mol.

[0454] According to some of any of the embodiments described herein, the low MW photocurable polymeric material has a poly(ethylene glycol) backbone featuring an average molecular weight (Mw) in range of from 1,000 to 4,000, or from 2,000 to 4,000, or from 3,000 to 4,000, or from 3,000 to 3,500, grams / mol, including any intermediate values and subranges therebetween or of about 3,400, grams / mol.

[0455] According to some of any of the embodiments described herein, the high MW photocurable polymeric material comprises a poly(alkylene glycol) polymeric backbone as described herein and terminates by two (or more) acrylate or methacrylate groups, as described herein.

[0456] According to some of these embodiments, an average molecular weight of the poly (alkylene glycol) polymeric backbone is higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol.

[0457] According to some of these embodiments, an average molecular weight of the poly (alkylene glycol) polymeric backbone ranges from 10,000 to 40,000, or from 10,000 to 30,000, or from 15,000 to 40,000, or from 15,000 to 30,000, or from 20,000 to 40,000, or from 20,000 to 30,000, or from 15,000 to 25,000, or from 20,000 to 25,000, grams / mol, including any intermediate values and subranges therebetween or about 20,000, grams / mol.

[0458] According to some of these embodiments, an average molecular weight of the poly(alkylene glycol) polymeric backbone ranges from 15,000 to 30,000, or from 20,000 to 30,000, or from 15,000 to 25,000, or from 20,000 to 25,000, grams / mol, including any intermediate values and subranges therebetween or about 20,000, grams / mol.

[0459] According to some of these embodiments, an average molecular weight of the poly(alkylene glycol) polymeric backbone is about 20,000, or about 21,000, or about 22,000, or about 23,000, grams / mol.

[0460] According to some of any of the embodiments described herein, the high MW photocurable polymeric material has a polymeric backbone which is a co-polymer of a high MW poly(alkylene glycol), e.g., a high MW poly(ethylene glycol) as described herein in any of the respective embodiments, and an additional polymeric material, that is, the polymeric backbone comprises backbone units of a poly(alkylene glycol) such as poly(ethylene glycol) and backbone units of an additional polymeric material, and the MW of the polymeric backbone is as described herein for the high MW polymeric material. In some of these embodiments, the co-polymeric material comprises one or more, preferably two or more photocurable groups as described herein, and in some embodiments, it is a linear co-polymer that terminates at each terminus by a photocurable group as described herein (e.g., acrylate or methacrylate).

[0461] According to some of any of these embodiments, the additional polymeric material is a biodegradable polymer other than poly(alkylene glycol).

[0462] According to some of any of these embodiments, the additional polymeric material is a biodegradable polymer other than poly (alkylene glycol), which features degradation rate higher than that of poly(ethylene glycol).

[0463] According to some of any of these embodiments, the additional polymeric material is a polyester or a copolymer thereof.

[0464] Exemplary polyesters include, without limitation, poly(lactic acid), poly(caprolactones), and poly(lactic acid-glycolic acid).

[0465] According to some of any of these embodiments, the additional polymeric material is or comprises poly(caprolactones), which is abbreviated herein as PCL.

[0466] According to some of any of these embodiments, the high MW photocurable polymeric material is a copolymer which is a block copolymer, as described herein.

[0467] According to some of any of these embodiments, the high MW photocurable polymeric material is a copolymer which is a block copolymer, as described herein, which comprises one or more blocks of a poly(alkylene glycol) such as poly(ethylene glycol) and one or more blocks of an additional polymeric material as described herein in any of the respective embodiments.

[0468] According to some of any of these embodiments, the high MW photocurable polymeric material is a copolymer which is a block copolymer, as described herein, which comprises one or more blocks of a poly(alkylene glycol) such as poly(ethylene glycol) and one or more blocks of a polyester as described herein in any of the respective embodiments.

[0469] According to some of any of these embodiments, the high MW photocurable polymeric material is a copolymer which is a block copolymer, as described herein, which comprises one or more blocks of a poly(alkylene glycol) such as poly(ethylene glycol) and one or more blocks of PCL.

[0470] According to some of any of these embodiments, the high MW photocurable polymeric material is a copolymer which is a block copolymer, as described herein, and comprises at least one of a poly(alkylene glycol) that features a high MW as described herein in any of the respective embodiments, that is, higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol, as described herein in any of the respective embodiments.

[0471] According to some of any of these embodiments, the high MW photocurable polymeric material is a copolymer which is a block copolymer, as described herein, and comprises a poly(alkylene glycol) that features a high MW as described herein in any of the respective embodiments, that is, higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol, as described herein in any of the respective embodiments, and one or more blocks of the additional polymeric material, for example, one or more blocks of PCL.

[0472] According to some of these embodiments, an average molecular weight of each of the one or more blocks of the additional polymeric material (e.g., PCL) is lower than 5,000, or lower than 3,000, or lower than 2,000, grams / mol.

[0473] According to some of these embodiments, an average molecular weight of each of the one or more blocks of the additional polymeric material (e.g., PCL) ranges from 500 to 5,000, or from, 500 to 4,000, or from 500 to 3,000, or from 500 to 2,000, or from 1,000 to 5,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, grams / mol, including any intermediate values and subranges therebetween or about 1,500, grams / mol.

[0474] According to some of any of these embodiments, the high MW photocurable polymeric material is a tri-block co-polymer that comprises the poly(alkylene glycol) polymeric backbone as a middle block having attached to its termini two additional blocks, each comprising a polymeric backbone of a biodegradable polymer other than poly(alkylene glycol) that terminates by a photocurable group.

[0475] According to some of the embodiments that relate to a tri-block co-polymer, an average MW of the poly (alkylene glycol) middle block higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol, as described herein in any of the respective embodiments, for example, is in a range of from 10,000 to 40,000, or from 10,000 to 30,000, or from 15,000 to 40,000, or from 15,000 to 30,000, or from 20,000 to 40,000, or from 20,000 to 30,000, or from 15,000 to 25,000, or from 20,000 to 25,000, or from 15,000 to 20,000, or from 18,000 to 22,000, grams / mol, including any intermediate values and subranges therebetween. According to some of these embodiments, an average MW of the poly(alkylene glycol) middle block is 15,000, or 16,000, or 17,000, or 18,000, or 19,000, or 20,000, or 21,000 or 22,000 or 23,000, grams / mol.

[0476] According to some of the embodiments that relate to a tri-block co-polymer, an average molecular weight of the one or more blocks of the additional polymeric material (e.g., PCL) is independently lower than 5,000, or lower than 3,000, or lower than 2,000, grams / mol.

[0477] According to some of the embodiments that relate to a tri-block co-polymer, an average molecular weight of the one or more blocks of the additional polymeric material (e.g., PCL) is independently is a range of from 500 to 5,000, or fro, 500 to 4,000, or from 500 to 3,000, or from 500 to 2,000, or from 1,000 to 5,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, grams / mol, including any intermediate values and subranges therebetween. According to some of the embodiments that relate to a block co-polymer, whenever it comprises two or more blocks other than poly(alkylene glycol), these blocks can be the same or different. When different, the blocks can differ in the average MW (Mw) and / or in the type of the polymeric material. In some embodiments, whenever a block co-polymer comprises two or more blocks other than poly(alkylene glycol), these blocks are the substantially the same.

[0478] According to some of any of the embodiments described herein, the high MW photocurable polymeric material is a triblock polymeric material that comprises a poly(alkylene glycol) polymeric backbone having a MW higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol, as described herein in any of the respective embodiments, and having attached to each of its termini a block of a polyester having a MW lower than 5,000, or lower than 3,000, or lower than 2,000, grams / mol and terminating by at least one (meth) acrylate group.

[0479] According to some of any of the embodiments described herein, the high MW photocurable polymeric material is a triblock polymeric material that comprises a poly(alkylene glycol) polymeric backbone having a MW in a range of from 10,000 to 30,000, or from 15,000 to 30,000, or from 20,000 to 30,000, or from 15,000 to 25,000, or from 20,000 to 25,000, or from 15,000 to 20,000, or from 18,000 to 22,000, grams / mol, including any intermediate values and subranges therebetween, and having attached to each of its termini a block of a polyester (e.g., PCL) having a MW independently is a range of from 500 to 3,000, or from 500 to 2,000, or from 1,000 to 2,000, grams / mol, including any intermediate values and subranges therebetween, and terminating by at least one (meth) acrylate group.

[0480] A tri-block copolymer of poly(ethylene glycol) and PCL can be readily synthesized by reacting poly(ethylene glycol) and e-caprolactone in the presence of a catalysts usable for polymerizing caprolactones (e.g., tin octanoate), to thereby obtain the tri-block copolymer that terminates by hydroxy groups, and reacting this tri-block copolymer with chloroacrylate or methacrylate under suitable conditions.

[0481] As exemplary structure of a high MW tri-block copolymer is as follows: wherein n and m are such that provide respective values of an average MW of the poly(ethylene glycol) (PEG) and of the PCL.

[0482] According to some of any of the embodiments described herein, the low MW photocurable polymeric material is a poly(alkylene glycol), such as poly(ethylene glycol) that terminates by two of the acrylate or methacrylate groups, preferably acrylate groups, and has an average molecular weight that ranges from 500 to 10,000, or from 500 to 8,000, or from 500 to 6,000, or from 500 to 5,000, or from 1,000 to 10,000, or from 2,000 to 10,000, or from 1,000 to 8,000, or from 2,000 to 8,000, or from 1,000 to 6,000, or from 2,000 to 6,000, or from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 6,000 or from 3,000 to 5,000 or from 3,000 to 4,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, or from 500 to 2,000 or from 500 to 1,500, grams / mol, including any intermediate values and subranges therebetween; and the high MW photocurable material is a triblock polymeric material that comprises a poly(alkylene glycol) such as poly(ethylene glycol) polymeric backbone having a MW higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol and having attached to each of its termini a block of a polyester having a MW lower than 5,000, or lower than 3,000, or lower than 2,000, grams / mol and terminating by the (meth)acrylate group.

[0483] According to some of any of the embodiments described herein, the low MW photocurable polymeric material is a poly(alkylene glycol), such as poly(ethylene glycol) that terminates by two of the acrylate or methacrylate groups, preferably acrylate groups, and has an average molecular weight that ranges from 1,000 to 5,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, or from 500 to 2,000 or from 500 to 1,500, grams / mol, including any intermediate values and subranges therebetween; and the high MW photocurable material is a triblock polymeric material that comprises a poly(alkylene glycol) such as poly(ethylene glycol) polymeric backbone having a MW higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol and having attached to each of its termini a block of a polyester having a MW lower than 5,000, or lower than 3,000, or lower than 2,000, grams / mol and terminating by the (meth)acrylate group.

[0484] According to some of any of the embodiments described herein, the low MW photocurable polymeric material is a poly(alkylene glycol), such as poly(ethylene glycol) that terminates by two of the acrylate or methacrylate groups, preferably acrylate groups, and has an average molecular weight that ranges from 500 to 10,000, or from 500 to 8,000, or from 500 to 6,000, or from 500 to 5,000, or from 1,000 to 10,000, or from 2,000 to 10,000, or from 1,000 to 8,000, or from 2,000 to 8,000, or from 1,000 to 6,000, or from 2,000 to 6,000, or from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 6,000 or from 3,000 to 5,000 or from 3,000 to 4,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, or from 500 to 2,000 or from 500 to 1,500, grams / mol, including any intermediate values and subranges therebetween; and the high MW photocurable material is a triblock polymeric material that comprises a poly(alkylene glycol) such as poly(ethylene glycol) polymeric backbone having a MW higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol, as described herein in any of the respective embodiments, and having attached to each of its termini a block of a polyester such as PCL having a MW in a range of from 500 to 3,000, or from 500 to 2,000, or from 1,000 to 2,000, grams / mol, including any intermediate values and subranges therebetween, and terminating by the (meth)acrylate group.

[0485] Exemplary formulations contemplated by the present invention for HC9 as the photoblocker and which include synthetic polymeric materials are provided in Tables B-I herein below. In one or more embodiments the carrier is an aqueous or hydrophilic carrier. In one or more embodiments, the carrier is a basic aqueous solution, a neutral aqueous solution e.g., PBS, or an acidic aqueous solution e.g., HC1. In some embodiments the carrier is an emulsion. In some embodiments the carrier is a microemulsion. In some embodiments the carrier is a nanoemulsion. In one or more embodiments the collagen is methacrylated to any degree (e.g., 50 % or 90 %). In one or more embodiments the collagen is lyophilized. According to a particular embodiment, the collagen is plant-derived recombinant collagen. In one or more embodiments, the photoinitiator is a salt of an acylphosphonate e.g., LAP or NAP.

[0486] Table C (corresponds to H2 in Examples)

[0487] Table D (corresponds to H3 in Examples) Table E (corresponds to H4)

[0488] Table F (corresponds to H5)

[0489] Table G (corresponds to H6 in Examples)

[0490] Table H (corresponds to H8 and H9) Table I (corresponds to H7)

[0491] Exemplary formulations contemplated by the present invention for HC9 as the photoblocker and which do not include synthetic polymeric materials are provided in Tables J-U hereinbelow. In one or more embodiments the carrier is an aqueous or hydrophilic carrier. In one or more embodiments, the carrier is a basic aqueous solution, a neutral aqueous solution e.g., PBS, or an acidic aqueous solution e.g. HC1. In some embodiments the carrier is an emulsion. In some embodiments the carrier is a microemulsion. In some embodiments the carrier is a nanoemulsion. In one or more embodiments the collagen is methacrylated to any degree (e.g., 50 % or 90 %). In one or more embodiments the collagen is lyophilized. According to a particular embodiment, the collagen is plant-derived recombinant collagen. In one or more embodiments, the photoinitiator is a salt of an acylphosphonate e.g., LAP or NAP. Table K (corresponds to H15 in Examples)

[0492] Table L (corresponds to H16 in Examples)

[0493] Table N (corresponds to H18 in Examples)

[0494] Table P (corresponds to H20 in Examples)

[0495] Table Q (corresponds to H21 in Examples) Table R (corresponds to H22 in Examples)

[0496] Table S (corresponds to H23 in Examples) Table T (corresponds to H24 in Examples)

[0497] Table U (corresponds to H25 in Examples)

[0498] Exemplary formulations contemplated by the present invention for GQ as the photoblocker are provided in Tables V-Y herein below. In one or more embodiments, GQ is dissolved in a polar solvent (e.g., ethanol). Each of formulations provided in the tables further comprises a carrier. In one or more embodiments, the carrier is a basic aqueous solution, a neutral aqueous solution e.g., PBS, or an acidic aqueous solution e.g., HCL. In some embodiments the carrier is an emulsion. In some embodiments the carrier is a microemulsion. In some embodiments the carrier is a nanoemulsion. In one or more embodiments the collagen is methacrylated to any degree (e.g. 50 % or 90 %). In one or more embodiments the collagen is lyophilized. According to a particular embodiment, the collagen is plant-derived recombinant collagen. In one or more embodiments, the photoinitiator is a salt of an acylphosphonate e.g., LAP or NAP.

[0499] Table V (corresponds to A in Examples)

[0500] Table W (corresponds to B in Examples)

[0501] Table X (corresponds to F24 in Examples) Table Y (corresponds to F26 in Examples)

[0502] Exemplary formulations contemplated by the present invention for TR as the photoblocker are provided in Tables Z-AB hereinbelow. Each of formulations provided in the tables further comprises a carrier. In one or more embodiments, the carrier is a basic aqueous solution, a neutral aqueous solution e.g., PBS or an acidic aqueous solution, e.g., HCL. In some embodiments the carrier is an emulsion. In some embodiments the carrier is a microemulsion. In some embodiments the carrier is a nanoemulsion. In one or more embodiments the collagen is methacrylated to any degree (e.g. 50 % or 90 %). In one or more embodiments the collagen is lyophilized. According to a particular embodiment, the collagen is plant-derived recombinant collagen. In one or more embodiments, the photoinitiator is a salt of an acylphosphonate e.g., LAP or NAP.

[0503] Table Z (corresponds to A A in Examples)

[0504] Table AA (corresponds to F23 in Examples)

[0505] Table AB (corresponds to F25 in Examples)

[0506] As mentioned, according to some of any of the embodiments described herein, the curable formulation further comprises a carrier and in some embodiments, the carrier is an aqueous carrier. In some embodiments the carrier is a hydrophilic carrier. In one or more embodiments, the carrier is a basic aqueous solution, a neutral aqueous solution e.g., PBS, or an acidic aqueous solution e.g., HC1. In some embodiments the carrier is an emulsion. In some embodiments the carrier is a microemulsion. In some embodiments the carrier is a nanoemulsion. In one or more embodiments the collagen is methacrylated to any degree (e.g., 50 % or 90 %). In one or more embodiments the collagen is lyophilized. According to a particular embodiment, the collagen is plant-derived recombinant collagen. In one or more embodiments, the photoinitiator is a salt of an acylphosphonate e.g., LAP or NAP. Exemplary formulations contemplated by the present invention for HC9 as the photoblocker are provided in Tables AC-AF hereinbelow. Each of formulations provided in the tables further comprises a carrier. In one or more embodiments, the carrier is a basic aqueous solution, a neutral aqueous solution e.g., PBS or an acidic aqueous solution, e.g., HCL. In some embodiments the carrier is an emulsion. In some embodiments the carrier is a microemulsion. In some embodiments the carrier is a nanoemulsion. In one or more embodiments the collagen is methacrylated to any degree (e.g. 50 % or 90 %). In one or more embodiments the collagen is lyophilized. According to a particular embodiment, the collagen is CollMa or GelMa. In one or more embodiments, the photoinitiator is a salt of an acylphosphonate e.g., LAP or NAP. Table AC (corresponds to H10 in Examples)

[0507] Table AD (corresponds to Hll in Examples)

[0508] Table AE (corresponds to H12 in Examples)

[0509] Table AF (corresponds to H13 in Examples)

[0510] The aqueous carrier can be water, a buffer featuring pH in a range of from about 2 to about

[0511] 10, or from about 2 to about 9, or from about 3 to about 9, or from about 3 to about 8, or from about 7 to about 10, or from about 8 to about 10, or from about 6 to about 8, or from about 3 to about 5, or from about 4 to about 6, or from about 2 to about 4, a basic aqueous solution, a neutral aqueous solution or an acidic aqueous solution.

[0512] The aqueous carrier can comprise salts and other water-soluble materials at varying concentrations. In some embodiments, a concentration of a salt in the carrier ranges from about 0.1 mM to about 0.2 M, or from about 0.1 mM to about 0.1 M, or from about 0.1 mM to about 100 mM, or from about 0.1 mM to about 50 mM, or from about 0.1 mM to about 20 mM, including an intermediate values and subranges therebetween.

[0513] In some embodiments, the aqueous carrier comprises salts at physiologically acceptable concentrations, such that the formulation features osmolarity around a physiological osmolarity.

[0514] In some embodiments the aqueous carrier comprises a phosphate salt, for example, a sodium phosphate monobasic (NaH PCh) and / or a sodium phosphate dibasic (sodium hydrogen phosphate; NaiHPCh). In some embodiments, the total concentration of the phosphate salt(s) in the formulation is about 0.1 M.

[0515] In some embodiments, the aqueous carrier comprises NaCl or any other physiologically acceptable salt.

[0516] In some embodiments, the aqueous carrier comprises a phosphate buffer and in some embodiments, the aqueous carrier comprises a phosphate buffer saline, which comprises sodium phosphate monobasic and / or sodium phosphate dibasic and NaCl or any other physiologically acceptable salt.

[0517] The phosphate buffer saline (PBS) can be a commercially available PBS (e.g., DPBS) or a custom-made buffer featuring a desirable pH and / or osmolarity.

[0518] In exemplary embodiments, the aqueous carrier comprises a phosphate buffer that comprises a phosphate sodium salt as described herein at a concentration of about 0.1M and NaCl at a concentration of from about 0.01 mM to about 200 mM, including any intermediate value and subranges therebetween.

[0519] Any other buffers are also usable in the context of the present embodiments.

[0520] In some of any of the embodiments described herein, the aqueous carrier comprises an acid.

[0521] In some embodiments, a concentration of the acid is lower than 100 mM, and can be, for example, of from 0.1 mM to 50 mM, or from 0.1 mM to 30 mM, or from 0.1 mM to 40 mM, or from 0.1 mM to 30 mM, or from 1 to 30 mM, or from 10 to 30 Mm, including any intermediate values and subranges therebetween.

[0522] The acid can be an inorganic acid (e.g., HC1) or an organic acid, preferably which is water soluble at the above-indicated concentrations (e.g., acetic acid). In some of any of the embodiments described herein, the aqueous carrier comprises a culturing medium. The culturing medium can be a commercially available culturing medium or a custom-made culturing medium. The culture medium can be any liquid medium which allows at least cell survival. Such a culture medium can include, for example, salts, sugars, amino acids and minerals in the appropriate concentrations and with various additives and those of skills in the art are capable of determining a suitable culture medium to specific cell types. Non-limiting examples of such culture medium include, phosphate buffered saline, DMEM, MEM, RPMI 1640, McCoy’s 5A medium, medium 199 and IMDM (available e.g., from Biological Industries, Beth Ha’emek, Israel; Gibco-Invitrogen Corporation products, Grand Island, NY, USA).

[0523] The culture medium may be supplemented with various antibiotics (e.g., Penicillin and Streptomycin), growth factors or hormones, specific amino acids (e.g., L-glutamin) cytokines and the like.

[0524] According to some of any of the embodiments described herein, the printing media (building material) in general or the curable formulation as described herein in particular further comprises a biological component or material other than the biocompatible curable material(s) (collectively referred to herein also as a biological material).

[0525] Biological components or materials that can be included in one or more curable (e.g., modeling material) formulations as described herein include cellular components, including, for example, culturing cells, and other cellular components such as cytokines, chemokines, growth factors; as well as other biological components such as proteins, agents that act to increase cell attachment, cell spreading, cell proliferation, cell differentiation and / or cell migration; an amino acid, peptides, polypeptides, proteins, DNA, RNA, lipids and / or proteoglycans.

[0526] Cells may comprise a heterogeneous population of cells or alternatively the cells may comprise a homogeneous population of cells. Such cells can be for example stem cells (such as embryonic stem cells, bone marrow stem cells, cord blood cells, mesenchymal stem cells, adult tissue stem cells), progenitor cells, or differentiated cells such as chondrocytes, osteoblasts, connective tissue cells (e.g., fibrocytes, fibroblasts and adipose cells), endothelial and epithelial cells. The cells may be naive or genetically modified.

[0527] According to one embodiment of this aspect of the present invention, the cells are mammalian in origin.

[0528] Furthermore, the cells may be of autologous origin or non- autologous origin, such as postpartum-derived cells (as described in U.S. Application Nos. 10 / 887,012 and 10 / 887,446). Typically, the cells are selected according to the desired application. Suitable proteins which can be used include, but are not limited to, extracellular matrix proteins [e.g., fibrinogen, collagen, fibronectin, vimentin, microtubule-associated protein ID, Neurite outgrowth factor (NOF), bacterial cellulose (BC), laminin and gelatin], cell adhesion proteins [e.g., integrin, proteoglycan, glycosaminoglycan, laminin, intercellular adhesion molecule (ICAM) 1, N-CAM, cadherin, tenascin, gicerin, RGD peptide and nerve injury induced protein 2 (ninjurin2)], growth factors [epidermal growth factor, transforming growth factor-a, fibroblast growth factor-acidic, bone morphogenic protein, fibroblast growth factor-basic, erythropoietin, thrombopoietin, hepatocyte growth factor, insulin-like growth factor-I, insulin-like growth factor-II, Interferon-P, platelet-derived growth factor, Vascular Endothelial Growth Factor and angiopeptin], cytokines [e.g., M-CSF, IF-lbeta, IE-8, beta-thromboglobulin, EMAP-II, G- CSF and IL- 10], proteases [pepsin, low specificity chymotrypsin, high specificity chymotrypsin, trypsin, carboxypeptidases, aminopeptidases, proline-endopeptidase, Staphylococcus aureus V8 protease, Proteinase K (PK), aspartic protease, serine proteases, cysteine proteases, metalloproteases, AD AMTS 17, tryptase-gamma, and matriptase-2] and protease substrates.

[0529] In addition, calcium phosphate materials, such as hydroxyapatite, for example, in a form of particles, can be used, including, but not limited to, nanoHA and nanoTCP. When the AM process involves dispensing the curable formulation through dispensing heads, the particles size should be compatible with the dispensing heads so as to avoid clogging.

[0530] Non-curable materials, other than the biological materials as described herein, that can be included in one or more curable (e.g., modeling material) formulations as described herein can be materials that impart a certain property to the formulation or to the hardened formulation or material and to the part of the object formed thereby. Such a property can be a physical property (e.g., an optical property such as transparency or opacity, color, a spectral property, heat resistance, electrical property and the like), or a mechanical or rheological property such as viscosity, elasticity, storage modulus, loss modulus, stiffness, hardness, and the like. Alternatively, or in addition, non-curable materials can be such that provide a biological function, for example, therapeutically active agents.

[0531] Exemplary non-curable materials include thixotropic agents, reinforcing agents, toughening agents, fillers, colorants, pigments, dye substances (e.g., as described herein), etc.

[0532] An exemplary non-curable material includes titanium dioxide.

[0533] An exemplary non-curable material includes oxidized cellulose.

[0534] According to some of any of the embodiments described herein, one or more of the curable (e.g., modeling material) formulations comprises hyaluronic acid. According to some of any of the embodiments described herein, one or more of the curable (e.g., modeling material) formulations comprises hyaluronic acid featuring a curable group as defined herein.

[0535] According to some of any of the embodiments described herein, one or more of the curable (e.g., modeling material) formulations comprises one or more biological components or materials such as, but not limited to, cells, growth factors, peptides, heparan sulfate and fibronectin.

[0536] According to some of any of the embodiments described herein, one or more of the curable (e.g., modeling material) formulations comprises one or more agents that modify a mechanical property of the formulation and / or the object, as described herein, such as, but not limited to, alginate, hyaluronic acid, fibrinogen, elastin, peptides and a thixotropic agent (e.g., Crystalline nano cellulose (CNC)), oxidized cellulose, titanium dioxide, Clay mineral and carbon nanotubes.

[0537] In some of any of the embodiments described herein the curable formulation further comprises a thixotropic agent, as defined herein.

[0538] Herein throughout, the term “thixotropic” describes a property of a fluidic compound or material that is reflected by a time-dependent shear- thinning, that is its viscosity is decreased in correlation with the time at which shear forces are applied, and returns back to its original value when application of shear forces is ceased. In some of the present embodiments, a thixotropic material or agent is such that exhibits or imparts a significant, e.g., at least 100 %, reduction in shear modulus under 50 % strain.

[0539] In some of any of the embodiments described herein, the curable formulation further comprises a gel-forming agent, for example, a hydrogel-forming agent as described herein.

[0540] In some of any of the embodiments described herein, the curable formulation further comprises a biological component or material as described herein.

[0541] In some of any of the embodiments described herein, the curable formulation further comprises one or more curable or non-curable materials as described herein in any of the respective embodiments.

[0542] According to some of any of the embodiments described herein, the 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.

[0543] According to some of any of the embodiments described herein the curable formulation further comprises one or more agents that modify a mechanical property of the formulation and / or the object, such as, but not limited to, alginate, hyaluronic acid, fibrinogen, elastin, peptides and a thixotropic agent (e.g., Crystalline nano cellulose (CNC)). In some of any of the embodiments described herein, all the curable materials in the building material are cured under the same curing condition, and are photocurable.

[0544] The following describes embodiments of additive manufacturing processes and methodologies for which the method as described herein can be employed.

[0545] According to an aspect of some embodiments of the present invention, there is provided a process (a method) of additive manufacturing (AM) of a three-dimensional object using the formulations described herein. According to embodiments of this aspect, the method is effected by sequentially forming a plurality of layers in a configured pattern corresponding to the shape of the object, thereby forming the object. According to some embodiments of this aspect, formation of each layer is effected by dispensing at least one uncured building material, and exposing the dispensed building material to a curing condition to thereby form a hardened (cured) material. According to some embodiments of this aspect, formation of each layer is effected by exposing a layer of uncured building material to a curing condition, and the method is effected by sequentially exposing, in a layer- wise manner, an uncured building material to a curing condition, whereby the exposure to the curing condition is effected in a configured pattern corresponding to the shape of the object.

[0546] Herein throughout, the phrase “building material” encompasses the phrases “uncured building material” or “uncured building material formulation” or “curable formulation” or “modeling material” or “ink“ or bioink” or “hydrogel” and collectively describes the materials that are used for sequentially forming the layers, as described herein. This phrase encompasses uncured materials which form the final object, namely, one or more uncured modeling material formulation(s), and optionally also uncured materials used to form a support, namely uncured support material formulations. The building material can also include non-curable materials that preferably do not undergo (or are not intended to undergo) any change during the process, for example, biological materials or components (other than a curable collagen as described herein) and / or other agents or additives as described herein.

[0547] The curable formulations described herein are suitable for printing with cells as they are advantageously free of short chain synthetic polymers. Such polymers, having a molecular weight of less than 1000MW, in their uncured form more readily diffuse into cells thus are cytotoxic. According to a particular embodiment, the formulations described herein are free or essentially free of short chain synthetic polymers having a molecular weight of less than 1000MW (e.g., PEGDMA700 or PEGDA700). According to some embodiments of the invention, the curable formulation is free of poly(caprolactones) (PCL) polymers. According to some embodiments of the invention, the curable formulation is free or essentially free of monomers (e.g., N-(2- Hydroxyethyl)acrylamide (HEAA)).

[0548] An uncured building material can comprise one or more modeling material formulations, and can be utilized such that different parts of the object are made upon hardening (e.g., curing) of different modeling formulations, and hence are made of different hardened (e.g., cured) modeling materials or different mixtures of hardened (e.g., cured) modeling materials.

[0549] The method of the present embodiments manufactures three-dimensional objects in a layerwise manner by forming a plurality of layers in a configured pattern corresponding to the shape of the object.

[0550] Each layer is formed by an additive manufacturing apparatus which scans a two- dimensional surface and patterns it. While scanning, the apparatus visits a plurality of target locations on the two-dimensional layer or surface, and decides, according to a pre-set algorithm, for each target location or a group of target locations, whether or not the target location or group of target locations is to be occupied by a building material, and which type of a building material is to be delivered thereto. The decision is made according to a computer image of the surface.

[0551] When the AM is by three-dimensional inkjet printing, an uncured building material, as defined herein, is dispensed from a dispensing head having a set of nozzles to deposit building material in layers on a supporting structure. The AM apparatus thus dispenses building material in target locations which are to be occupied and leaves other target locations void. The apparatus typically includes a plurality of dispensing heads, each of which can be configured to dispense a different building material (for example, different modeling material formulations, each containing a different biological component; or each containing a different curable material; or each containing a different concentration of a curable material, and / or different support material formulations). Thus, different target locations can be occupied by different building materials (e.g., a modeling formulation and / or a support formulation, as defined herein).

[0552] The final three-dimensional object is made of the hardened or cured modeling material or a combination of hardened modeling materials or a combination of hardened modeling material / s and support material / s or modification thereof (e.g., following curing). All these operations are well-known to those skilled in the art of additive manufacturing (also known as solid freeform fabrication).

[0553] In some exemplary embodiments of the invention an object is manufactured by dispensing a building material that comprises two or more different modeling material formulations, each modeling material formulation from a different dispensing head of the AM apparatus. The modeling material formulations are optionally and preferably deposited in layers during the same pass of the dispensing heads. The modeling material formulations and / or combination of formulations within the layer are selected according to the desired properties of the object.

[0554] An exemplary process according to some embodiments of the present invention starts by receiving 3D printing data corresponding to the shape of the object. The data can be received, for example, from a host computer which transmits digital data pertaining to fabrication instructions based on computer object data, e.g., in a form of a Standard Tessellation Language (STL) or a StereoLithography Contour (SLC) format, Virtual Reality Modeling Language (VRML), Additive Manufacturing File (AMF) format, Drawing Exchange Format (DXF), Polygon File Format (PLY), Digital Imaging and Communications in Medicine (DICOM) or any other format suitable for Computer-Aided Design (CAD).

[0555] The process continues by dispensing the building material as described herein in layers, on a receiving medium, using one or more dispensing (e.g., printing) heads, according to the printing data.

[0556] The dispensing can be in a form of droplets, or a continuous stream, depending on the additive manufacturing methodology employed and the configuration of choice.

[0557] The receiving medium can be a tray of a printing system, or a supporting article or medium made of, or coated by, a biocompatible material, such as support media or articles commonly used in bioprinting, or a previously deposited layer.

[0558] In some embodiments, the receiving medium comprises a sacrificial hydrogel or other biocompatible material as a mold to embed the printed object, and is thereafter removed by chemical, mechanical or physical (e.g., heating or cooling) means. Such sacrificial hydrogels can be made of, for example, a Pluronic material or of Gelatin.

[0559] Once the uncured building material is dispensed on the receiving medium according to the 3D data, the method optionally and preferably continues by hardening the dispensed formulation(s). In some embodiments, the process continues by exposing the deposited layers to a curing condition. Preferably, the curing condition is applied to each individual layer following the deposition of the layer and prior to the deposition of the previous layer.

[0560] As used herein throughout, the term “curing” describes a process in which a formulation is hardened. The hardening of a formulation typically involves an increase in viscosity of the formulation and / or an increase in a storage modulus of the formulation (G’). In some embodiments, a formulation which is dispensed as a liquid becomes solid or semi-solid (e.g., gel) when hardened. A formulation which is dispensed as a semi-solid (e.g., soft gel) becomes solid or a harder or stronger semi-solid (e.g., strong gel) when hardened. The term “curing” as used herein encompasses, for example, polymerization of monomeric and / or oligomeric materials and / or cross-linking of polymeric chains (either of a polymer present before curing or of a polymeric material formed in a polymerization of the monomers or oligomers). The product of a curing reaction is therefore typically a polymeric material and / or a cross-linked material. This term, as used herein, encompasses also partial curing, for example, curing of 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 formulation, in addition to curing of 100 % of the formulation.

[0561] Herein, the phrase “a condition that affects curing” or “a condition for inducing curing”, which is also referred to herein interchangeably as “curing condition” or “curing inducing condition” describes a condition which, when applied to a formulation that contains a curable material, induces a curing as defined herein. Such a condition can include, for example, application of a curing energy, as described hereinafter to the curable material(s), and / or contacting the curable material(s) with chemically reactive components such as catalysts, co-catalysts, and activators.

[0562] When a condition that induces curing comprises application of a curing energy, the phrase “exposing to a curing condition” and grammatical diversions thereof means that the layers are exposed to the curing energy and the exposure is typically performed by applying a curing energy to the layers.

[0563] A “curing energy” typically includes application of radiation (irradiation) or application of heat.

[0564] The radiation can be electromagnetic radiation (e.g., ultraviolet or visible light), or electron beam radiation, or ultrasound radiation or microwave radiation, depending on the materials to be cured. The application of radiation (or irradiation) is effected by a suitable radiation source. For example, an ultraviolet or visible or infrared or Xenon or mercury or lamp, or LED source, can be employed, as described herein.

[0565] A curable material or system that undergoes curing upon exposure to radiation is referred to herein interchangeably as “photopolymerizable” or “photoactivatable” or “photocurable”.

[0566] When the curing energy comprises heat, the curing is also referred to herein and in the art as “thermal curing” and comprises application of thermal energy. Applying thermal energy can be effected, for example, by heating a receiving medium onto which the layers are dispensed or a chamber hosting the receiving medium, as described herein. In some embodiments, the heating is effected using a resistive heater.

[0567] In some embodiments, the heating is effected by irradiating the dispensed layers by heatinducing radiation. Such irradiation can be effected, for example, by means of an IR lamp or Xenon lamp, operated to emit radiation onto the deposited layer. In some embodiments, heating is effected by infrared radiation applied by a ceramic lamp, for example, a ceramic lamp that produces infrared radiation of from about 3 pm to about 4 pm, e.g., about 3.5 pm.

[0568] A curable material or system that undergoes curing upon exposure to heat is referred to 5 herein as “thermally-curable” or “thermally-activatable” or “thermally-polymerizable”.

[0569] In some of any of the embodiments described herein, hardening the formulation(s) comprises exposing the formulation to a curing condition which is irradiation (illumination), as described herein.

[0570] In some embodiments, the exposure to a curing condition (irradiation) is for a short time period, for example, a time period of less than 3 minutes, less than 300 seconds, for example, of 10 from 10 seconds to 240 seconds, or from 10 seconds to 120 seconds, or from 10 seconds to 60 seconds, or from 10 seconds to 30 seconds, or from 10 seconds to 15 seconds, including an intermediate values and subranges therebetween.

[0571] In some embodiments, exposing to the curing condition (irradiation) is for a time period that ranges from 1 second to 120 second or from 1 second to 20 seconds, including an intermediate 15 values and subranges therebetween.

[0572] In some embodiments, the irradiation is at wavelength within the UV-vis range.

[0573] In some embodiments, the irradiation is at wavelength in a range of from about 300 to about 800, or from about 300 to about 600, or from about 300 to about 500, or from about 350 to about 450, nm, including any intermediate values and subranges therebetween. In exemplary 20 embodiments, the irradiation is at 405 nm. In exemplary embodiments, the irradiation is at 385 nm. In exemplary embodiments, the irradiation is at 365 nm.

[0574] In some embodiments, the irradiation is at a power intensity level that ranges from about 1 to about 150, or from about 1 to about 130, or from about 1 to about 100, or from about 10 to about 150, or from about 10 to about 130, or from about 10 to about 100, or from about 50 to about 150, 25 or from about 50 to about 130, or from about 50 to about 100, or from about 1 to about 50 or from about 1 to about 30 or from about 1 to about 20, from about 1 to about 15, from about 1 to about 9, from about 1 to about 10 mW / cm2, including any intermediate values and subranges therebetween.

[0575] According to one preferred embodiment, the irradiation is at a power intensity level that 30 ranges from about 1 to about 30 mW / cm2, or from about 10 to about 30 mW / cm2. According to one preferred embodiment, the irradiation is at a power intensity level that ranges from about 5 to about 15 mW / cm2, or from about 20 to about 30. According to one preferred embodiment, the irradiation is at a power intensity level that ranges from about 10 to about 30 mW / cm2. According to one preferred embodiment, the irradiation is at a power intensity level of about 10 or about 27 mW / cm2-

[0576] In some of any of the embodiments described herein, the exposure time, controls the amount of energy delivered by the light source to the printed layer. In some embodiments, the exposure energy is at a level that ranges from about 1 to about 250, 1 to about 200, 1 to about 150, or from about 1 to about 130, or from about 1 to about 100, or from about 10 to about 150, or from about 10 to about 130, or from about 10 to about 100, or from about 50 to about 150, or from about 50 to about 130, or from about 50 to about 100, or from about 1 to about 50, or from about 1 to about 40, or from about 1 to about 30 or from about 1 to about 20, from about 1 to about 15, from about 1 to about 9, from about 1 to about 10 mJ / cm2, including any intermediate values and subranges therebetween.

[0577] In some of any of the embodiments described herein, the exposure time, controls the amount of energy delivered by the light source to the printed layer. In some embodiments, the exposure energy is at a level that ranges from about 100 to about 600, 200 to about 600, or from about 300 to about 600, or from about 400 to about 600, or from about 500 to about 600, or from about 100 to about 200, or from about 100 to about 300, 100 to about 400 or from about 100 to about 500, or from about 450 to about 530, or from about 450 to about 600, or from about 450 to about 550, or from about 500 to about 640, or from about 500 to about 630 or from about 500 to about 620, from about 500 to about 550, from about 550 to about 600, from about 300 to about 400 mJ / cm2, including any intermediate values and subranges therebetween.

[0578] In some embodiments, the irradiation of the base layer is ranges from about 100 to about 400 mJ / cm2

[0579] In some embodiments, printing is controlled by one or more parameters comprising exposure energy, exposure time, layer thickness, concentration of the photo initiator or the concentration photo blocker.

[0580] In some of any of the embodiments described herein, the method further comprises exposing the cured modeling material formulation(s) either before or after removal of a support material formulation, if such has been included in the building material, to a post-treatment condition. The post-treatment condition is typically aimed at further hardening the cured modeling material(s). In some embodiments, the post-treatment hardens a partially-cured formulation to thereby obtain a completely cured formulation.

[0581] In some embodiments, the post-treatment (post cure) is effected by exposure to heat or radiation, as described in any of the respective embodiments herein. Some embodiments contemplate the fabrication of an object by dispensing different formulations from different dispensing heads. These embodiments provide, inter alia, the ability to select formulations from a given number of formulations and define desired combinations of the selected formulations and their properties.

[0582] According to the present embodiments, the spatial locations of the deposition of each formulation with the layer are defined, either to effect occupation of different three-dimensional spatial locations by different formulations, or to effect occupation of substantially the same three- dimensional location or adjacent three-dimensional locations by two or more different formulations so as to allow post deposition spatial combination of the formulations within the layer.

[0583] The present embodiments thus enable the deposition of a broad range of material combinations, and the fabrication of an object which may consist of multiple different combinations of modeling material formulations, in different parts of the object, according to the properties desired to characterize each part of the object.

[0584] A system utilized in additive manufacturing may include a receiving medium and one or more dispensing heads. The receiving medium can be, for example, a fabrication tray that may include a horizontal surface to carry the material dispensed from the printing head. In some embodiments, the receiving medium is made of, or coated by, a biocompatible material, as described herein.

[0585] The dispensing head may be, for example, a printing head having a plurality of dispensing nozzles arranged in an array of one or more rows along the longitudinal axis of the dispensing head. The dispensing head may be located such that its longitudinal axis is substantially parallel to the indexing direction.

[0586] The additive manufacturing system may further include a controller, such as a microprocessor to control the AM process, for example, the movement of the dispensing head according to a pre-defined scanning plan (e.g., a CAD configuration converted to a Standard Tessellation Language (STL) format and programmed into the controller). The dispensing head may include a plurality of jetting nozzles. The jetting nozzles dispense material onto the receiving medium to create the layers representing cross sections of a 3D object.

[0587] In addition to the dispensing head, there may be a source of curing energy, for curing the dispensed building material. The curing energy is typically radiation, for example, UV radiation or heat radiation. Alternatively, there may be means for providing a curing condition other than electromagnetic or heat radiation, for example, means for cooling the dispensed building material or for contacting it with a reagent that promotes curing. Additionally, the AM system may include a leveling device for leveling and / or establishing the height of each layer after deposition and at least partial solidification, prior to the deposition of a subsequent layer.

[0588] According to the present embodiments, the additive manufacturing method described herein is for bioprinting a biological object.

[0589] As used herein, "bioprinting" means practicing an additive manufacturing process while utilizing one or more curable formulation(s) that comprise(s) biological components, as described herein, via methodology that is compatible with an automated or semi-automated, computer-aided, additive manufacturing system as described herein (e.g., a bioprinter or a bioprinting system).

[0590] In some embodiments, the bioprinting comprises sequential formation of a plurality of layers of the uncured building material in a configured pattern, preferably according to a three- dimensional printing data, as described herein. At least one, and preferably most or all, of the formed layers (before hardening or curing) comprise(s) one or more biological component(s) as described herein (e.g., a curable Collagen as described herein). Optionally, at least one of the formed layers (before hardening or curing) comprises one or more non-biological curable materials, and / or non-curable biological or non-biological components, preferably biocompatible materials which do not interfere (e.g., adversely affect) with the biological and / or structural features of the biological components (e.g., collagen) in the printing medium and / or bio-ink. According to some embodiments described herein, the bioprinting process further comprises exposing at least a portion of the formed layers to a curing condition which comprises curing energy, for example, light energy (irradiation, illumination).

[0591] In some of any of the embodiments described herein, the bioprinting method is configured to effect formation of the layers under conditions that do not significantly affect structural and / or functional properties of the biological components in the bioink composition.

[0592] In some embodiments, a bioprinting system for effecting a bioprinting process / method as described herein is configured so as to allow formation of the layers under conditions that do not significantly affect structural and / or functional properties of the biological components in the bioink.

[0593] In some of any of the embodiments described herein, the additive manufacturing (e.g., bioprinting) process and system are configured such that the process parameters (e.g., temperature, shear forces, shear strain rate) do not interfere with (do not substantially affect) the functional and / or structural features of the biological components.

[0594] According to some of the present embodiments, the additive manufacturing is of a three- dimensional object featuring, in at least a portion thereof, a collagen-based material, and comprises dispensing at least one modeling material formulation to sequentially form a plurality of layers in a configured pattern corresponding to a shape of the object, wherein for at least a portion of the layers, the dispensing is of one or more modeling material formulation(s) that comprise the curable formulation as described herein in any of the respective embodiments and any combination thereof.

[0595] According to some of any of the embodiments described herein, the process further comprises exposing at least a portion of the dispensed layers to a curing condition which comprises curing energy, for example, light energy (irradiation, illumination).

[0596] According to some of any of the embodiments described herein, for at least a portion of the layers, the dispensing is further of a modeling material formulation that comprises an agent that modifies a mechanical and / or rheological and / or physical property of the formulation and / or of a respective portion of the object.

[0597] According to some of any of the embodiments described herein, or at least a portion of the layers, the formation of the layers or dispensing is further of a modeling material formulation that comprises a biological material other than the biocompatible photocurable material (e.g., curable collagen) as described herein.

[0598] According to some of any of the embodiments described herein, the dispensing or forming of layers is at a temperature that ranges from -10 to 50 °C, or from -4 to 50 °C, or from -4 to 37 °C or from 4 to 20 °C or from 4 to 25 °C or from 10 to 20 °C or from 10 to 25 °C, or from 20 to 30 °C or from 20 to 25 °C or from 10 to 30 °C or from 20 to 37 °C or from 25 to 37 °C or from 4 to 37 °C including an intermediate values and subranges therebetween. In some embodiments, the temperature is at least 10 °C, or at least 15 °C or of at least 20 °C, or at least 22, °C or at least 25 °C, or at least 30 °C or at room temperature or at a temperature of 37 °C.

[0599] In some of any of the embodiments described herein, the additive manufacturing process (the bioprinting) is performed at a temperature of at least 10 °C, or of at least 20 °C, for example, at a temperature that ranges from about 10 to about 40 °C, preferably from about 10 °C to 37 °C, or from about 20 °C to 37 °C, or from about 20 °C to about 30 °C, or from about 20 °C to about 28 °C, or from about 20 °C to about 25 °C, including any intermediate values and subranges therebetween, or at room temperature, or at 37 °C.

[0600] In some of any of the embodiments described herein, the above-indicated temperatures / temperature ranges are the temperatures at which the building material (e.g., at least a modeling material formulation that comprises a biological component as described herein) are dispensed, that is, a temperature of a dispensing head in the AM system and / or a temperature at which the modeling material formulation is maintained during layer formation or prior to passing in the dispensing head and / or a temperature at which the modeling material formulation is maintained in the vat prior to and / or during curing.

[0601] In some of any of the embodiments described herein, the AM process is performed without cooling the AM system (e.g., without cooling the dispensing heads and / or a modeling material formulation and / or vat), to a temperature below room temperature, e.g., a temperature lower than 25 °C or lower than 20 °C or lower than 10 °C, or lower than 5 °C (e.g., 4 °C).

[0602] In some of any of the embodiments described herein, the AM system is devoid of means for cooling the system or a part thereof (e.g., means for cooling the dispensing heads and / or the modeling material formulation and / or the vat), to a temperature below room temperature, e.g., 25 °C or lower than a temperature lower than 20 °C or lower than 10 °C, or lower than 5 °C (e.g., 4 °C).

[0603] In some of any of the embodiments described herein, the additive manufacturing process (bioprinting) is performed while applying a shear force that does not adversely affect structural and / or functional properties of biological components (e.g., cells). Applying the shear force can be effected by passing the building material (e.g., at least a modeling material formulation that comprises a biological component as described herein) through the dispensing head, and is to be regarded also as subjecting the building material to shear force.

[0604] Some embodiments of the present invention allow to perform AM bioprinting processes under conditions that do not affect the functional and / or structural features of biological components included in the bio-ink (e.g., at low shear force and room temperature or a physiological temperature), while maintaining the required fluidity (a viscosity that imparts fluidity, e.g., lower than 10,000 centipoises or lower than 5,000 centipoises, lower than 2,000 lower than 1000 or lower than 500 centipoises), and while further maintaining the curability of the building material.

[0605] The following describes exemplary AM bioprinting methodologies that are usable in the context of embodiments of the present invention.

[0606] A bioprinting method and a corresponding system can be any of the methods and systems known in the art for performing additive manufacturing, and exemplary such systems and methods are described hereinabove. A suitable method and system can be selected upon considering its printing capabilities, which include resolution, deposition speed, scalability, bio-ink compatibility and ease-of-use.

[0607] Exemplary suitable bioprinting systems usually contain a dispensing system (either equipped with temperature control module or at ambient temperature), and stage (a receiving medium), and a movement along the x, y and z axes directed by a CAD-CAM software. A curing source (e.g., a light or heat source) which applies a curing energy (e.g., by applying light or heat radiation) or a curing condition to the deposition area (the receiving medium) so as to promote curing of the formed layers and / or a humidifier, can also be included in the system. There are printers that use multiple dispensing heads to facilitate a serial dispensing of several materials.

[0608] Generally, bioprinting can be effected using any of the known techniques for additive manufacturing. The following lists some exemplary additive manufacturing techniques, although any other technique is contemplated.

[0609] 3D Inkjet printing:

[0610] 3D Inkjet printing is a common type of 3D printer for both non-biological and biological (bioprinting) applications. Inkjet printers use thermal or acoustic forces to eject drops of liquid onto a substrate, which can support or form part of the final construct. In this technique, controlled volumes of liquid are delivered to predefined locations, and a high-resolution printing with precise control of (1) ink drops position, and (2) ink volume, which is beneficial in cases of microstructureprinting or when small amounts of bioreactive agents or drugs are added, is received. Inkjet printers can be used with several types of ink, for example, comprising multiple types of biological components and / or bioactive agents. Furthermore, the printing is fast and can be applied onto culture plates.

[0611] A bioprinting method that utilizes a 3D inkjet printing system can be operated using one or more bio-ink modeling material formulations as described herein, and dispensing droplets of the formulation(s) in layers, on the receiving medium, using one or more inkjet printing head(s), according to the 3D printing data.

[0612] PolyJet or MJP printing:

[0613] Other technologies suitable for bioprinting applications involving UV-cured materials, thanks to their precision, multi-material capabilities, and effective UV curing process are MultiJet Printing (MJP) or PolyJet technology which jets photopolymer droplets and cures them using UV light after each layer is deposited. While MJP / PolyJet shares some commonalities with 3D inkjet printing due to their use of inkjet technology for material deposition, they differ significantly in the types of materials used, curing processes, and typical applications.

[0614] Two-Photon Polymerization (2PP) and some forms of Selective Laser Sintering (SLS) or / Selective Laser Melting (SLM):

[0615] Additional printing technologies which rely on photoblockers to absorb light are Two- Photon Polymerization (2PP) and some forms of Selective Laser Sintering (SLS) or / Selective Laser Melting (SLM). These technologies may use different wavelengths of light, often in the near-infrared or visible spectrum, and do not always rely on UV for curing. In 2PP a femtosecond laser induces polymerization within a photoresist. Photoblockers in the resin are key to enabling this process, which allows for extremely fine resolution, even at the nanoscale. While SLS and SLM technologies are typically used for powdered materials, there are variants where photopolymers or polymer-powder composites are used, requiring photoblockers to absorb laser energy and cause the sintering or melting of the material. In one embodiment, the 2PP, SLS or SLM rely on UV for curing.

[0616] Stereolithography (SLA) and Digital Light Processing (DLP):

[0617] SLA and DLP are additive manufacturing technologies in which an uncured building material in a bath or vat is converted into hardened material(s), layer by layer, by selective curing using a light source while the uncured material is later separated / washed from the hardened material. SLA is widely used to create models, prototypes, patterns, and production parts for a range of industries including for Bioprinting. DLP differs from laser-based SLA in that DLP uses a projection of ultraviolet (UV) light (or visible light) from a digital projector to flash a single image of the layer across the entire uncured material at once. One of the key components of DLP is a digital micromirror device (DMD) chip, which is typically composed of an array of reflective aluminum micromirrors that redirect incoming light from the UV source to project an image of a designed pattern. For achieving a high-resolution structure, parameters such as the curing time of each layer, layer thickness, and intensity of the UV light should be tuned, for example, by controlling the concentration and types of the curable materials, the photoabsorber and / or the photoinitiator.

[0618] Laser-assisted printing:

[0619] Laser-assisted printing technique, in the version adopted for 3D bioprinting, is based on the principle of laser- induced forward transfer (LIFT), which was developed to transfer metals and is now successfully applied to biological materials. The device consists of a laser beam, a focusing system, an energy absorbing / converting layer and a biological material layer (e.g., cells and / or hydrogel) and a receiving substrate. A laser assisted printer operates by shooting a laser beam onto the absorbing layer which converts the energy into a mechanical force which drives tiny drops from the biological layer onto the substrate. A light source is then utilized to cure the material on the substrate.

[0620] Laser assisted printing is compatible with a series of viscosities and can print mammalian cells without affecting cell viability or cell function. Cells can be deposited at a density of up to 108cells / ml with microscale resolution of a single cell per drop. In one embodiment, Laser-assisted printing technique relies on UV for curing. Electrospinning:

[0621] Electrospinning is a fiber production technique, which uses electric force to draw charged threads of polymer solutions, or polymer melts. In one embodiment, the electrospinning further includes a light-induced reaction. In one embodiment, the electrospinning further relies on UV for curing.

[0622] Direct Ink Writing (DIW):

[0623] DIW uses high-viscosity photocurable inks (e.g., resins with ceramic, polymer, or nanoparticle fillers). Ink is extruded as filaments, then cured either continuously (with a UV lamp near the nozzle) or after deposition (layer by layer). Allows much higher filler content (ceramics, composites, hydrogels) than inkjet. Resolution is lower than inkjet because extrusion filaments are thicker than droplets.

[0624] According to some of any of the embodiments described herein, the additive manufacturing (bioprinting) is or comprises digital light processing (DLP), as described herein.

[0625] According to an aspect of some embodiments of the present invention, there is provided a process (or method) of additive manufacturing using DLP technology, wherein a three-dimensional object featuring, in at least a portion thereof, a biological or a biocompatible material is prepared. In some embodiments, a 3D digital model is created or obtained through the use of computer-aided design (CAD) software, as described herein. In an embodiment, specialized slicing software is employed, slicing the 3D model into distinct cross-sectional layers, or "slices" as described herein, with varying layer thickness and slicing parameters. In accordance with some embodiments, a curable formulation is chosen with specific regard to the desired properties and characteristics of the final object, and the vat is prepared. In some embodiments, the DLP 3D printing process is implemented by following the method steps as described herein in any of the respective embodiments. In an embodiment, the digital light projector, through the projection of highly detailed images onto the curable formulation surface, ensures precision and accuracy in layer-by- layer additive printing. According to some embodiments, post-processing procedures are carried out after the completion of printing, including rinsing to remove excess, uncured formulation and optional post-curing (e.g., by application of electromagnetic irradiation and / or heat). According to some embodiments, post-processing procedures are carried out after the completion of printing including drying and / sterilization. In some embodiments, post-processing procedures do not significantly affect mechanical, biological, structural and / or functional properties of the biological components in the bio-ink or the bioprinted object.

[0626] According to some of any of the embodiments described herein, the additive manufacturing is effected by sequentially exposing in a layer-wise manner a curable formulation as described herein in any of the respective embodiments and any combination thereof to a curing condition such as UV irradiation, as described herein in any of the respective embodiments and any combination thereof, and the exposing is performed in a configured pattern corresponding to the shape of the object (in accordance with a pre-determined computerized software as described herein), such that the curable formulation is hardened in each layer at locations exposed to the curing condition, as typically performed in DLP processes. In some of any of these embodiments, the thickness of each layer, the level of irradiation (energy dose [mJ / cm2]), power intensity [mW / cm2] and / or the time of exposing [sec] each layer to irradiation are determined or manipulated so as to provide the optimal resolution for a curable formulation of choice. According to some of any of the embodiments described herein, the level of irradiation and the time of exposing to irradiation is as described herein in any of the respective embodiments and any combination thereof.

[0627] Herein throughout, in the context of bioprinting, the term “object” describes a final product of the additive manufacturing which comprises, in at least a portion thereof, a biological component. This term refers to the product obtained by a bioprinting method as described herein, after removal of the support material, if such has been used as part of the uncured building material.

[0628] The term "object" as used herein throughout refers to a whole object or a part thereof.

[0629] In the context of the present embodiments, the object comprises in at least a portion thereof a biocompatible material obtained from the biocompatible photocurable material(s) as described herein, for example, a collagen-based material. In some embodiments, the object is in a form of a scaffold, for example, a hydrogel scaffold, as described herein.

[0630] In some of any of the embodiments described herein, the object is in a form of a tissue or organ. Such an object can be formulated in accordance with a respective 3D printing data of a desired organ or tissue, using, in addition to the curable collagen as described herein, additional curable materials and biological materials as described herein.

[0631] In some embodiments, the object is an implantable object. In some embodiments, the object is an artificial skin. In some embodiments, the object is an artificial tissue (e.g., connective tissue, or muscle tissue such as cardiac tissue and pancreatic tissue). Examples of connective tissues include, but are not limited to, cartilage (including, elastic, hyaline, and fibrocartilage), adipose tissue, reticular connective tissue, embryonic connective tissues (including mesenchymal connective tissue and mucous connective tissue), tendons, ligaments, and bone.

[0632] In some embodiments, the object is usable in, or is for use in, constructing an artificial organ or tissue. The object can comprise hardened materials formed of the one or more photocurable curable materials as described herein in any of the respective embodiments, biological components or materials, as described herein in any of the respective embodiments, and / or non-curable materials as described herein in any of the respective embodiments.

[0633] The scaffolds may be administered to subjects in need thereof for the regeneration of tissue such as connective tissue, muscle tissue such as cardiac tissue and pancreatic tissue, or any other tissue, or can be used for research purposes.

[0634] The films can be used to construct biomedical devices such as, for example, collagen membranes for hemodialysis.

[0635] According to some embodiments, films or scaffolds can be used in cell cultures.

[0636] The phrase "cell culture" or "culture" as used herein refers to the maintenance of cells in an artificial, e.g., an in vitro environment. It is to be understood, however, that the term "cell culture" is a generic term and may be used to encompass the cultivation not only of individual prokaryotic (e.g., bacterial) or eukaryotic (e.g., animal, plant and fungal) cells, but also of tissues, organs, organ systems or whole organisms.

[0637] In some embodiments, the films or scaffolds can be used in a wound healing process.

[0638] The object of the present embodiments comprises a myriad of other uses including, but not limited to, in the treatment of diseases such as interstitial cystitis, scleroderma, and rheumatoid arthritis cosmetic surgery, as a healing aid for bum patients, as a wound-healing agent, as a dermal filler, for spinal fusion procedures, for urethral bulking, in duraplasty procedures, for reconstruction of bone and a wide variety of dental, orthopedic and surgical purposes.

[0639] The object may form a part of an article-of-manufacturing such as, for example, a medical device, including an implantable medical device (e.g., a breast implant or a dermal filler). The object may form a part of an article-of-manufacturing such as, for example, a scaffold for cosmetic or aesthetic use, including an implantable scaffold (e.g., a breast implant for use in soft tissue augmentation).

[0640] According to an aspect of some embodiments of the present invention, there is provided a kit that comprises components of a curable formulation as described herein in any of the respective embodiments. Typically, the kit will comprise the photoblocker, the photoinitiator, and collagen (as described herein above). According to a particular embodiment, the collagen is plant-derived recombinant collagen. In one embodiment, the collagen is lyophilized. In one or more embodiments the collagen is methacrylated to any degree (e.g. 50 % or 90 %). According to some of any of the embodiments described herein, the kit is identified for use, or is usable, as a modeling material formulation for additive manufacturing (e.g., bioprinting) of an object as described herein in any of the respective embodiments.

[0641] According to some of any of the embodiments described herein, the kit further comprises an aqueous carrier, as described herein in any of the respective embodiments. In some embodiments, each of the components of the curable formulation and the aqueous carrier are packaged individually within the kit.

[0642] Alternatively, the kit includes instructions to prepare a modeling material formulation as described herein, by mixing curable formulation with the aqueous carrier.

[0643] The kit may further comprise other components that can be included in a curable formulation or a modeling material formulation(s) as described herein in any of the respective embodiments and any combination thereof.

[0644] The kit may further comprise instructions how to use the curable formulation in an additive manufacturing process as described herein.

[0645] The kit may comprise a curable formulation without a photoblocker, and instructions to use a method as described herein for selecting a suitable photoblocker and an amount thereof, to be added to the curable formulation,

[0646] According to an aspect of some embodiments of the present invention there is provided a process of additive manufacturing a three-dimensional object featuring, in at least a portion thereof, a biological or a biocompatible material, which comprises sequentially exposing a curable formulation that comprises photoblocker or a dye substance as described herein in any of the respective embodiments, to irradiation at a wavelength of from 300 to 800 nm, in layerwise manner, and in a configured pattern corresponding to a shape of the object.

[0647] In some embodiments, the exposing is for time period that ranges from 1 second to 120 seconds or from 1 second to 20 seconds for each layer.

[0648] In some embodiments, the irradiation is at a wavelength that ranges from 300 to 600 nm, or from 300 to 500 nm, or from 350 to 450 nm, or is 385 nm.

[0649] The exposure (energy) mJ / cm2, is the power intensity (mW / cm2) multiplied by the time of exposure (seconds), wherein the energy of irradiation is equal to the light source power intensity multiplied by the time of exposure.

[0650] In some embodiments, when the intensity is 27 mW / cm2, the exposure time ranges from 1 to 60 sec to obtain an energy of irradiation which ranges from 27 to 1620 mJ / cm2. In some embodiments, the intensity is 27 mW / cm2, and the exposure time for printing the foundation layers ranges from 15 to 60, or from 20 to 60 seconds, or from 30 to 60 seconds, or from 40 to 60 seconds, or from 44 to 60 seconds. In some embodiments, the intensity is 27 mW / cm2, and the exposure time for printing the foundation layers ranges from 15 to 30 seconds. In some embodiments, the intensity is 27 mW / cm2, and the exposure time for printing the scaffold layers ranges from 1 to 12 seconds, 2 to 12 seconds, or from 3 to 12 seconds, or from 4 to 12 seconds, or from 5 to 12 seconds, 1 to 16, or from 2 to 16 seconds, or from 3 to 16 seconds, or from 4 to 16 seconds, or from 5 to 16 seconds, 1 to 20, or from 2 to 20 seconds, or from 3 to 20 seconds, or from 4 to 20 seconds, or from 5 to 20 seconds. In some embodiments, the intensity is 27 mW / cm2, and the exposure time for printing the scaffold layers ranges from 10 to 15 seconds.

[0651] In some embodiments, when the exposure duration is 44 seconds, the intensity is fixed at 27 mW / cm2to obtain an energy of irradiation of approximately 1200 mJ / cm2. In some embodiments, the exposure duration is 10 sec, and the intensity for printing the foundation layers ranges from 1 to 120 mW / cm2. In some embodiments, the exposure duration is 10 sec, and the intensity for printing the scaffold layers ranges from 1 to 90 mW / cm2.

[0652] In some embodiments, the exposure duration is 44 sec, and the intensity is fixed at 27 mW / cm2. In other embodiments, the exposure duration may be 20 sec with intensity ranging from 1 to 60 mW / cm2, or 40 sec with intensity ranging from 1 to 30 mW / cm2, or 80 sec with intensity ranging from 1 to 15 mW / cm2, or 100 sec with intensity ranging from 1 to 12 mW / cm2. In some embodiments, the exposure duration is 5 sec with intensity ranging from 1 to 240 mW / cm2. In some embodiments, the exposure duration is 10 sec with intensity ranging from 1 to 120 mW / cm2. In some embodiments, the exposure duration is 20 sec with intensity ranging from 1 to 60 mW / cm2. In some embodiments, the exposure duration is 40 sec with intensity ranging from 1 to 30 mW / cm2. In some embodiments, the exposure duration is 50 sec with intensity ranging from 1 to 24 mW / cm2.

[0653] In some embodiments, the intensity is 40 mW / cm2and the exposure duration ranges from 1 to 30 sec. In some embodiments, the intensity is 27 mW / cm2and the exposure duration is 44 sec. In some embodiments, the intensity is 20 mW / cm2and the exposure duration ranges from 1 to 60 sec. In some embodiments, the intensity is 10 mW / cm2and the exposure duration ranges from 1 to 120 sec. In some embodiments, the intensity is 5 mW / cm2and the exposure duration ranges from 1 to 240 sec.

[0654] In some embodiments, the energy of irradiation ranges from 27 to 1620 mJ / cm2In some embodiments, the energy of irradiation ranges from 100 to 1600 mJ / cm2, 120 to 1200 mJ / cm2, 150 to 1350 mJ / cm2, 200 to 1400 mJ / cm2, 300 to 1500 mJ / cm2In some embodiments, the energy of irradiation ranges from 1100 to 1300 mJ / cm2, or from 1150 to 1250 mJ / cm2, or specifically at approximately 1200 mJ / cm2. In some embodiments, the energy of irradiation for printing the foundation layers ranges from 1000 to 1600 mJ / cm2. In some embodiments, the energy of irradiation for printing the scaffold layers ranges from 1000 to 1300 mJ / cm2. In some embodiments, the energy of irradiation for printing the foundation layers ranges from 1100 to 1400 mJ / cm2In some embodiments, the energy of irradiation for printing the scaffold layers ranges from 1000 to 1200 mJ / cm2

[0655] In some embodiments, the intensity of the printer’s light source ranges from 1 to 150 mW / cm2. In some embodiments, the intensity of the printer’s light source ranges from 1 to 120 mW / cm2, 1 to 100 mW / cm2, 1 to 80 mW / cm2, 1 to 60 mW / cm2, 1 to 50 mW / cm2, 1 to 40 mW / cm2, 1 to 30 mW / cm2, 1 to 27 mW / cm2, or 1 to 20 mW / cm2.

[0656] In some embodiments, when the intensity is 10mW / cm2, the exposure time ranges from 1 to 60 sec to obtain an energy of irradiation which ranges is from 10 to 600 mJ / cm2. In some embodiments, the intensity is 10mW / cm2, the exposure time for printing the foundation layers ranges from 15 to 60 or from 20 to 60 seconds, or from 30 to 60 seconds, or from 40 to 60 seconds, or from 50 to 60 seconds. In some embodiments, the intensity is 10mW / cm2, and the exposure time for printing the foundation layers ranges or from 15 to 30seconds. In some embodiments, the intensity is 10mW / cm2, and the exposure time for printing the for printing the scaffold layers ranges from 1 to 12 seconds, 2 to 12 seconds, or from 3 to 12 seconds, or from 4 to 12 seconds, or from 5 to 12 seconds 1 to 16, or from 2 to 16 seconds, or from 3 to 16 seconds, or from 4 to 16 seconds, or from 5 to 16 seconds, 1 to 20, or from 2 to 20 seconds, or from 3 to 20 seconds, or from 4 to 20seconds, or from 5 to 20 seconds. In some embodiments, the intensity is 10mW / cm2, and the exposure time for printing the scaffold layers ranges or from 10 to 15 seconds.

[0657] In some embodiments, when the exposure duration is lOsec, the intensity ranges from 1 to 60mW / cm2 to obtain the energy of irradiation which ranges from 10 to 600 mJ / cm2. In some embodiments, the exposure duration is lOsec, and the intensity for printing the foundation layers ranges from 1 to 23mW / cm2. In some embodiments, the exposure duration is lOsec, and the intensity for printing the scaffold layers ranges from 1 to 15mW / cm2.

[0658] In some embodiments, the exposure duration is lOsec, the intensity ranges from 1 to 40mW / cm2. In some embodiments, the exposure duration is 20sec, the intensity ranges from 1 to

[0659] 20mW / cm2. In some embodiments, the exposure duration is 40sec, the intensity ranges from 1 to

[0660] 10mW / cm2. In some embodiments, the exposure duration is 80sec, the intensity ranges from 1 to

[0661] 5mW / cm2. In some embodiments, the exposure duration is lOOsec, the intensity ranges from 1 to

[0662] 4mW / cm2.

[0663] In some embodiments, the exposure duration is 5sec, the intensity ranges from 1 to 40mW / cm2. In some embodiments, the exposure duration is lOsec, the intensity ranges from 1 to 20mW / cm2. In some embodiments, the exposure duration is 20sec, the intensity ranges from 1 to 10mW / cm2. In some embodiments, the exposure duration is 40sec, the intensity ranges from 1 to 5mW / cm2. In some embodiments, the exposure duration is 50sec, the intensity ranges from 1 to 4mW / cm2.

[0664] In some embodiments, the intensity is 40mW / cm2 and the exposure duration ranges from 1 to lOsec. In some embodiments, the intensity is 20mW / cm2, and the exposure duration ranges 1 to 20sec. In some embodiments, the intensity is 10mW / cm2, and the exposure duration is ranges 1 to 40sec. In some embodiments, the intensity is 5mW / cm2, and the exposure duration ranges 1 to 80sec. In some embodiments, the intensity is 40mW / cm2 and the exposure duration ranges from 1 to 5sec. In some embodiments, the intensity is 20mW / cm2, and the exposure duration is ranges 1 to lOsec. In some embodiments, the intensity is 10mW / cm2, and the exposure duration is ranges 1 to 20sec. In some embodiments, the intensity is 5mW / cm2, and the exposure duration is ranges 1 to 40sec.

[0665] In some embodiments, the energy of irradiation ranges from 10 to 600 mJ / cm2. In some embodiments, the energy of irradiation ranges from 10 to 500 mJ / cm2, 10 to 400mJ / cm2, 10 to 300 mJ / cm2, 10 to 200 mJ / cm2, 10 to 160 mJ / cm2, 10 to 150 mJ / cm2, 10 to 140 mJ / cm2, 10 to 130 mJ / cm2, 10 to 120 mJ / cm2, 10 to 110mJ / cm2 , 10 to 100mJ / cm2 ,10 to 90mJ / cm2, 10 to 80 mJ / cm2, 10 to 70 mJ / cm2, 10 to 60mJ / cm2, 10 to 50mJ / cm2, 10 to 40mJ / cm2, 10 to 30mJ / cm2 , 10 to 20mJ / cm2. In some embodiments, the energy of irradiation ranges from 110 to 300 mJ / cm2, 110 to 200mJ / cm2, 120 to 300 mJ / cm2, 120 to 200 mJ / cm2, 120 to 160 mJ / cm2, 110 to 250 mJ / cm2, 110 to 240 mJ / cm2, 110 to 230 mJ / cm2, 110 to 220 mJ / cm2, 110 to 210mJ / cm2 , 115 to 200mJ / cm2 ,110 to 190mJ / cm2, 110 to 180 mJ / cm2, 110 to 170 mJ / cm2, 160 to 260mJ / cm2, 160 to 250mJ / cm2, 160 to 240mJ / cm2, 160 to 230mJ / cm2 , 160 to 220mJ / cm2, 160 to 210mJ / cm2, 180 to 220 mJ / cm2, 190 to 220 mJ / cm2, 200 to 220mJ / cm2, 180to 210mJ / cm2, 180 to 210mJ / cm2, 190 to 210mJ / cm2 , 190 to 200mJ / cm2.In some embodiments, the energy of irradiation for printing the foundation layers ranges from 150 to 600 mJ / cm2. In some embodiments, the energy of irradiation for printing the scaffold ranges from 100 to 150 mJ / cm2. In some embodiments, the energy of irradiation for printing the foundation layers ranges from 150 to 300 mJ / cm2. In some embodiments, the energy of irradiation for printing the scaffold ranges from 110 to 150 mJ / cm2.

[0666] In some embodiments, the intensity of the printer’s light source ranges from 1 to 150 mW / cm2. In some embodiments, the intensity of the printer’s light source ranges from 1 to 100 mW / cm2, 1 to 90 mW / cm2, 1 to 80 mW / cm2, 1 to 70 mW / cm2, Ito 60 mW / cm2,lto 50 mW / cm2, 1 to 40 mW / cm2, 1 to 30 mW / cm2, 1 to 20 mW / cm2, 1 to 10 mW / cm2, 1 to 9 mW / cm2, 1 to 8 mW / cm2, 1 to 7 mW / cm2, 1 to 6 mW / cm2. In some embodiments, a thickness of exposed layers, a level of the irradiation (energy), power intensity, a time of the exposing, and / or an amount of the photoblocker are determined while executing a method as described herein in any of the respective embodiments, for example, while determining a curing kinetics and / or printing resolution in the presence of a selected photoblocker.

[0667] In some embodiments, the additive manufacturing is DLP.

[0668] As used herein the term “about” refers to ± 10 % or ± 5 %.

[0669] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0670] The term “consisting of’ means “including and limited to”.

[0671] The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0672] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

[0673] Throughout this application, various embodiments of this 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 should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0674] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0675] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0676] Herein throughout, whenever “centipoise” or “Cp” is indicated, the corresponding Pa- second value (1 Pa- second = 1,000 centipoise) is encompassed.

[0677] Herein throughout, whenever the phrase “weight percent”, or “% by weight” or “% wt.”, is indicated in the context of embodiments of a formulation (e.g., a modeling formulation, a curable formulation, a bioink composition), it is meant weight percent of the total weight of the respective uncured formulation.

[0678] Herein throughout, the phrase “linking moiety” or “linking group” describes a group that connects two or more moieties or groups in a compound. A linking moiety is typically derived from a bi- or tri-functional compound, and can be regarded as a bi- or tri-radical moiety, which is connected to two or three other moieties, via two or three atoms thereof, respectively.

[0679] Exemplary linking moieties include a hydrocarbon moiety or chain, optionally interrupted by one or more heteroatoms, as defined herein, and / or any of the chemical groups listed below, when defined as linking groups.

[0680] When a chemical group is referred to herein as “end group” it is to be interpreted as a substituent, which is connected to another group via one atom thereof.

[0681] Herein throughout, the term “hydrocarbon” collectively describes a chemical group composed mainly of carbon and hydrogen atoms. A hydrocarbon can be comprised of alkyl, alkene, alkyne, aryl, and / or cycloalkyl, each can be substituted or unsubstituted, and can be interrupted by one or more heteroatoms. The number of carbon atoms can range from 2 to 30, and is preferably lower, e.g., from 1 to 10, or from 1 to 6, or from 1 to 4. A hydrocarbon can be a linking group or an end group.

[0682] As used herein, the term “amine” describes both a -NR’R” group and a -NR'- group, wherein R’ and R" are each independently hydrogen, alkyl, cycloalkyl, aryl, as these terms are defined hereinbelow.

[0683] The amine group can therefore be a primary amine, where both R’ and R” are hydrogen, a secondary amine, where R’ is hydrogen and R” is alkyl, cycloalkyl or aryl, or a tertiary amine, where each of R’ and R” is independently alkyl, cycloalkyl or aryl.

[0684] 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, Nthiocarbamate, Othiocarbamate, urea, thiourea, Ncarbamate, Ocarbamate, C-amide, Namide, guanyl, guanidine and hydrazine. The term “amine” is used herein to describe a -NR'R" group in cases where the amine is an end group, as defined hereinunder, and is used herein to describe a -NR'- group in cases where the amine is a linking group or is or part of a linking moiety.

[0685] The term "alkyl" describes a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 30, or 1 to 20 carbon atoms. Whenever a numerical range; e.g., " 1-20", is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. The alkyl group may be substituted or unsubstituted. Substituted alkyl may have one or more substituents, whereby each substituent group 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, Nthiocarbamate, Othiocarbamate, urea, thiourea, Ncarbamate, Ocarbamate, C-amide, N-amide, guanyl, guanidine and hydrazine.

[0686] The alkyl group can be an end group, as this phrase is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking group, as this phrase is defined hereinabove, which connects two or more moieties via at least two carbons in its chain. When the alkyl is a linking group, it is also referred to herein as “alkylene” or “alkylene chain”.

[0687] Alkene and Alkyne, as used herein, are an alkyl, as defined herein, which contains one or more double bond or triple bond, respectively.

[0688] The term "cycloalkyl" describes an all-carbon monocyclic ring or fused rings (z.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. Examples include, without limitation, cyclohexane, adamantine, norbomyl, isobomyl, and the like. The cycloalkyl group may be substituted or unsubstituted. Substituted cycloalkyl may have one or more substituents, whereby each substituent group 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, Nthiocarbamate, Othiocarbamate, urea, thiourea, Ncarbamate, Ocarbamate, Camide, Namide, guanyl, guanidine and hydrazine. The cycloalkyl group can be an end group, as this phrase is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking group, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof. The term "heteroalicyclic" describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino, oxalidine, and the like.

[0689] The heteroalicyclic may be substituted or unsubstituted. Substituted heteroalicyclic may have one or more substituents, whereby each substituent group 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, Nthiocarbamate, Othiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine. The heteroalicyclic group can be an end group, as this phrase is defined hereinabove, where it is attached to a single adjacent atom, or a linking group, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof.

[0690] The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (z.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. The aryl group may be substituted or unsubstituted. Substituted aryl may have one or more substituents, whereby each substituent group 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, Nthiocarbamate, Othiocarbamate, urea, thiourea, Ncarbamate, Ocarbamate, C-amide, N-amide, guanyl, guanidine and hydrazine. The aryl group can be an end group, as this term is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking group, as this term is defined hereinabove, connecting two or more moieties at two or more positions thereof.

[0691] The term "heteroaryl" describes a monocyclic or fused ring (z.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted. Substituted heteroaryl may have one or more substituents, whereby each substituent group 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, Nthiocarbamate, Othiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine. The heteroaryl group can be an end group, as this phrase is defined hereinabove, where it is attached to a single adjacent atom, or a linking group, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof. Representative examples are pyridine, pyrrole, oxazole, indole, purine and the like.

[0692] The term "halide" and “halo” describes fluorine, chlorine, bromine or iodine.

[0693] The term “haloalkyl” describes an alkyl group as defined above, further substituted by one or more halide.

[0694] The term "carbonyl" or "carbonate" as used herein, describes a -C(=O)-R’ end group or a -C(=O)- linking group, as these phrases are defined hereinabove, with R’ as defined herein.

[0695] The term "thiocarbonyl" as used herein, describes a -C(=S)-R’ end group or a -C(=S)- linking group, as these phrases are defined hereinabove, with R’ as defined herein.

[0696] The term “oxo” as used herein, describes a (=0) group, wherein an oxygen atom is linked by a double bond to the atom (e.g., carbon atom) at the indicated position.

[0697] The term “thiooxo” as used herein, describes a (=S) group, wherein a sulfur atom is linked by a double bond to the atom (e.g., carbon atom) at the indicated position.

[0698] The term “oxime” describes a =N-0H end group or a =N-O- linking group, as these phrases are defined hereinabove.

[0699] The term “hydroxyl” describes a -OH group.

[0700] The term "alkoxy" describes both an -O-alkyl and an -O-cycloalkyl group, as defined herein. The term alkoxide describes -R’O“ group, with R’ as defined herein.

[0701] The term "aryloxy" describes both an -O-aryl and an -O-heteroaryl group, as defined herein.

[0702] The term "thiohydroxy" or “thiol” describes a -SH group. The term “thiolate” describes a -S’ group.

[0703] The term "thioalkoxy" describes both a -S-alkyl group, and a -S-cycloalkyl group, as defined herein.

[0704] The term "thioaryloxy" describes both a -S-aryl and a -S-heteroaryl group, as defined herein.

[0705] The “hydroxyalkyl” is also referred to herein as “alcohol”, and describes an alkyl, as defined herein, substituted by a hydroxy group.

[0706] The term “acyl halide” describes a -(C=O)R"" group wherein R"" is halide, as defined hereinabove. The term “carboxylate” as used herein encompasses C-carboxylate and O-carboxylate.

[0707] The term “Ccarboxylate” describes a -C(=O)-OR’ end group or a -C(=0)-0 linking group, as these phrases are defined hereinabove, where R’ is as defined herein.

[0708] The term “Ocarboxylate” describes a -OC(=O)R’ end group or a -OC(=O) linking group, as these phrases are defined hereinabove, where R’ is as defined herein.

[0709] A carboxylate can be linear or cyclic. When cyclic, R’ and the carbon atom are linked together to form a ring, in C-carboxylate, and this group is also referred to as lactone. Alternatively, R’ and O are linked together to form a ring in O-carboxylate. Cyclic carboxylates can function as a linking group, for example, when an atom in the formed ring is linked to another group.

[0710] The term “thiocarboxylate” as used herein encompasses C-thiocarboxylate and O- thiocarboxylate.

[0711] The term “Cthiocarboxylate” describes a -C(=S)-OR’ end group or a -C(=S)-0 linking group, as these phrases are defined hereinabove, where R’ is as defined herein.

[0712] The term “Othiocarboxylate” describes a -OC(=S)R’ end group or a -OC(=S) linking group, as these phrases are defined hereinabove, where R’ is as defined herein.

[0713] A thiocarboxylate can be linear or cyclic. When cyclic, R’ and the carbon atom are linked together to form a ring, in C-thiocarboxylate, and this group is also referred to as thiolactone. Alternatively, R’ and O are linked together to form a ring in O-thiocarboxylate. Cyclic thiocarboxylates can function as a linking group, for example, when an atom in the formed ring is linked to another group.

[0714] The term “carbamate” as used herein encompasses N-carbamate and O-carbamate.

[0715] The term “N-carbamate” describes an R”OC(=O)-NR’- end group or a OC(=O)-NR’- linking group, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0716] The term “O-carbamate” describes an -OC(=O)-NR’R” end group or an -OC(=O)-NR’ linking group, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0717] A carbamate can be linear or cyclic. When cyclic, R’ and the carbon atom are linked together to form a ring, in O-carbamate. Alternatively, R’ and O are linked together to form a ring in N-carbamate. Cyclic carbamates can function as a linking group, for example, when an atom in the formed ring is linked to another group.

[0718] The term “carbamate” as used herein encompasses N-carbamate and O-carbamate..

[0719] The term “thiocarbamate” as used herein encompasses N-thiocarbamate and O- thiocarbamate.

[0720] The term “O-thiocarbamate” describes a OC(=S)NR’R” end group or a OC(=S)NR’ linking group, as these phrases are defined hereinabove, with R’ and R” as defined herein. The term “N-thiocarbamate” describes an R”OC(=S)NR’- end group or a OC(=S)NR’- linking group, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0721] Thiocarbamates can be linear or cyclic, as described herein for carbamates.

[0722] The term “dithiocarbamate” as used herein encompasses S -dithiocarbamate and N- dithiocarbamate.

[0723] The term “S -dithiocarbamate” describes a SC(=S)NR’R” end group or a SC(=S)NR’ linking group, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0724] The term “N-dithiocarbamate” describes an R”SC(=S)NR’- end group or a SC(=S)NR’- linking group, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0725] The term "urea", which is also referred to herein as “ureido”, describes a -NR’C(=O)- NR”R’ ’ ’ end group or a -NR’C(=O)-NR”- linking group, as these phrases are defined hereinabove, where R’ and R” are as defined herein and R'" is as defined herein for R' and R".

[0726] The term “thiourea”, which is also referred to herein as “thioureido”, describes a -NR’- C(=S)-NR”R”’ end group or a -NR’-C(=S)-NR” linking group, with R’, R” and R’” as defined herein.

[0727] The term “amide” as used herein encompasses C-amide and N-amide.

[0728] The term “C-amide” describes a -C(=O)-NR’R” end group or a -C(=O)-NR’- linking group, as these phrases are defined hereinabove, where R’ and R” are as defined herein.

[0729] The term “N-amide” describes a R’C(=O)-NR”- end group or a R’C(=O)-N- linking group, as these phrases are defined hereinabove, where R’ and R” are as defined herein.

[0730] An amide can be linear or cyclic. When cyclic, R’ and the carbon atom are linked together to form a ring, in C-amide, and this group is also referred to as lactam. Cyclic amides can function as a linking group, for example, when an atom in the formed ring is linked to another group.

[0731] As used herein, the term “alkylene glycol” describes a -O-[(CR’R”)Z-O]y-R”’ end group or a -O-[(CR’R”)Z-O]y- linking group, with R’, R” and R’” being as defined herein, and with z being an integer of from 1 to 10, preferably, from 2 to 6, more preferably 2 or 3, and y being an integer of 1 or more. Preferably R’ and R” are both hydrogen. When z is 2 and y is 1, this group is ethylene glycol. When z is 3 and y is 1, this group is propylene glycol. When y is 2-4, the alkylene glycol is referred to herein as oligo(alkylene glycol). When y is higher than 4, it is a poly(alkylene glycol). Capped poly(alkylene glycol) has R’” which is other than hydrogen and which can be, for example, an alkyl (e.g., lower alkyl), a carbonyl, and like moieties.

[0732] Exemplary methods and protocols, as described herein, are described in the Examples section that follows, although any other methods and protocols known in the art are contemplated. A person skilled in the art would readily recognized how to perform these steps, typically using conventional and / or widely applied protocols known in the art.

[0733] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0734] 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.

[0735] EXAMPLES

[0736] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0737] EXAMPLE 1

[0738] Selection criteria of photoblockers

[0739] A photoblocker is an important component in formulations usable in 3D-bioprinting (formulations comprising a biological component such as ECM proteins, e.g., collagen), particularly when printing in methods requiring high resolution (e.g., DLP method). Inclusion of a suitable photoblocker may enhance the printing resolution, and may also beneficially affect the hardening or curing kinetics, the cross-linking degree and swelling. Crosslinking reaction must proceed at a controlled rate - fast enough to enable rapid layer solidification and maintain printing efficiency, yet slow enough to prevent over-curing, which can lead to loss of detail or pore closure. Properly tuned kinetics ensures uniform curing across the entire layer and between successive layers, supporting consistent mechanical performance and dimensional stability.

[0740] The present inventors have thus looked for dye substances that are suitable for inclusion in the formulation as photoblockers.

[0741] Dyes listed in Table 1A were evaluated to see if they meet one or more of the following criteria:

[0742] (i) Non-toxicity, preferably biocompatibility (optional registration as generally safe for use, for example, in the FDA Inactive Ingredient Guide (IIG). Dye substances that are chemically stable under UV irradiation do not undergo decomposition thus maintain lack of toxicity post curing.

[0743] (ii) Dye substances having solubility of at least 0.05 mg / mL in the selected carrier at room temperature, or of sufficient solubility that it is able to function as a photoblocker in a curable formulation. For example, the upper limit suitable for use in a curable formulation which still provides a desirable resolution (e.g., by adjusting the amount of the dye substance and / or amount of photoinitiator and / or suitable process parameters such as irradiation energy dose, irradiation time).

[0744] (iii) Dye substances that exhibit absorbance at selected wavelength(s) within the UV- vis range (300-800 nm).

[0745] (iv) Dye substances that are stable under UV irradiation at the selected wavelength(s) with and without a selected photoinitiator. Dye substances that do not undergo decomposition into non-absorbing substances post irradiation at printing absorbance spectra (or that do not shift their wavelength) are deemed suitable.

[0746] (v) Dye substances that are compatible in the curable formulation to be utilized in the 3D bioprinting. Compatible means maintaining physical and chemical properties of the formulation in terms of, for example, solubility of all the components, including the dye substance, in the formulation, homogeneity, viscosity of the formulation, and maintaining chemical stability of the components (e.g., chemical inertness of the dye substances towards each and / or all the components in the formulation), in the presence of the dye substance. In an example, solubility of all the components in the formulation upon introducing the dye substance to the formulation at a concentration sufficient for exhibiting absorbance at a selected wavelength is indicative of formulation compatibility.

[0747] (vi) Dye substances that when added to a curable formulation result in a controlled curing or crosslinking kinetics. The controlled rate (time to G' >100 Pa) results in printed model having an acceptable resolution for the formulation of interest. The kinetic parameter for evaluation, which is associated with the steep increase in G' values in response to illumination, is time to reach G' >100 Pa. Shorter time to reach G' > 100 Pa reflects faster curing kinetics. For example, F25 based on TR as the dye substance had a time to G' [Sec] of less than 11 second to reach G' >100 Pa did not adversely effect on the storage modulus G' = 14 + / - 6 [KPa].

[0748] (vii) Dye substances that enhance the printing resolution at least at the Z axis, compared to formulations without the dye substance, and preferably also do not adversely affect the printing resolution at the XY plane. (viii) Dye substances that enhance or do not significantly adversely affect a mechanical property such as, for example, tensile strength, tensile strain, elastic modulus, etc., of the hardened material or printed model.

[0749] (ix) Dye substances that do not adversely affect the biocompatibility of the hardened material or printed model.

[0750] (x) Dye substances that are chemically stable that do not undergo oxidation or degradation. This can be determined, for example by analyzing change of color or chromatography in the presence of oxygen or polar solvents such as water.

[0751] (xi) Dye substances that are colorless or light colored for improved patient compliance and aesthetics.

[0752] (xii) Dye substances that are easily washed out of the hardened material.

[0753] EXAMPLE 2

[0754] Experimental Materials, Formulations and Methods Dye substance candidates listed in Table 1A feature varying structural features, spanning from metal salts to porphyrins, through varying mono-cyclic and multi-cyclic substances were tested.

[0755] Table 1A A summary of materials used in Examples 3-9 and their corresponding manufactures is provided in Table IB

[0756] Table IB

[0757] A summary of formulations used in studies described in Examples 3-9 comprising HC9, GQ, TR, and MC as photoblockers is provided in Tables 2-6, Table 7, Table 8 and Table 9 respectively below.

[0758] Table 2: HC9 formulations comprising single polymer PEGDA 3.4K

[0759] Table 3: HC9 formulations comprising PEGDA1K to PCL-PEG-PCL 24K PEGDMA 1:1

[0760] Table 4: HC9 formulations comprising PEGDA1K to PCL-PEG-PCLDMA OR 23K OR PEGDMA 20K (1:3) Table 5: HC9 formulations comprising GelMA OR COLLMA AND PEGDA 3.4K

[0761] Table 6: HC9 polymer free formulations

[0762] Table 7

[0763] Table 8 Table 9 The following exemplary experimental methods were used in studies described in Examples 3-6, conducted while implementing the designed methodology for identifying dye substances suitable for use as photoblockers in a photocurable formulation that comprises photocurable biological material such as collagen or GelMA and optionally one or more curable synthetic biocompatible polymers, an aqueous carrier and an acyl phosphine oxide-type photoinitiator such as LAP or NAP, as defined herein in any of the respective embodiments.

[0764] Method 1 Solubility

[0765] Tested photoblockers were dissolved in 10 mM HC1 or DDW at a final concentration of 0.1-10 mM, optionally with sonication (Example 3 for PB 1-14).

[0766] In an alternative procedure, 5 mg / mL of a tested photoblocker were added to a 10 mM HC1 solution at room temperature and the solution was stirred. Solubility was assessed by visual inspection after 5 minutes. A clear solution and lack of precipitation are indicative that the photoblocker is soluble in 10 mM HC1.

[0767] When a tested photoblocker was found to be insoluble at the tested concentration, the upper solubility limit (the maximal concentration in which the dye substance is soluble and provides satisfactory resolution for intended use) was determined. If the resolution model of tested photoblocker was determined unsatisfactory at such upper solubility limit, the solubility of the photoblocker was evaluated in other solvents. For example, the upper solubility limit of 3- -D- Glucosylquercetin was determined as 0.5 % wt (5mg / ml 3-0-D-Glucosylquercetin in ethanol).

[0768] Method 2 UV-absorbance

[0769] DDW was used as a blank for solutions diluted in DDW, and 10 mM HC1 was used as a blank for solutions diluted in 10 mM HC1. 2 pL of each tested solution was put in the nanodrop and the absorbance between 190 nm to 850 nm was read (see Example 3 PB 1-12).

[0770] In an alternative procedure, a 10 mM HC1 solution was used as a reference sample; and a 10 mM dye solution was prepared in 1.5 mL of 10 mM HC1 and if solubility therein was confirmed absorbance was tested. If solubility in 10 mM HC1 was not confirmed, solutions were further diluted and absorbance was read at the maximal concentration at which a tested dye substance was soluble. In case such maximal concentration resulted in insufficient resolution other solvents were used to dissolve the tested dye (e.g., PB-23 was dissolved in ethanol) and such solvents were used as the reference sample.

[0771] UV absorbance was measured with a spectrophotometer, upon transferring 1 mL of each solution to a disposable cuvette. Results were evaluated using an absorbance-intensity curve that was plotted as a function of wavelength, km ax of the photoblocker was extracted from the curve as well as the absorbance intensity at 365, 385 and / or 405 nm. Photoblockers that exhibited absorbance at 365, 385 and / or 405 nm were selected for use with DLP printers, see Example 3 PB 13-14, Example 5 PB-23 and Example 6 PB-22.

[0772] Method 3 Stability under UV irradiation

[0773] UV absorbance measurements were performed for 1 mL 10 mM of a HC1 solution containing each tested photoblocker at a concentration of 10 mM or 0.5 % by weight, or lower concentration (the maximal concentration at which a tested dye substance is soluble in case it is insoluble), alone, or in combination with 0.5 % by weight the photoinitiator NAP (before and after irradiation) at wavelength used for printing 365nm at 100 % intensity 20sec (see Example 3 PB 1- PB-14) or 385 nm at 100 % intensity 10 or 20 sec (see Example 3 PB- 15-21, Example 5 PB-23 and Example 6 PB-22). Absorbance measurements were conducted as described in method 2.

[0774] The Beer-Lambert Law assumes a linear relationship between absorbance and concentration. Maintaining compliance with Beer- Lambert Law ensures accurate, reproducible measurements in spectrophotometry. Spectrophotometers have a reliable absorbance range, often between 0.1 to 1.0 AU. Outside this range, the readings are inaccurate. At high concentrations, molecules are so close together that they interact, affecting how they absorb light.

[0775] As absorbance values for PB- 17, PB-22, PB-23 were in the non-linear Beer- Lambert range, to ensure accurate results, the samples were re-prepared at lower concentrations (solutions were diluted using a solvent) and photoabsorbance was reevaluated. Initially, the absorbance of each PB in solution was measured. Subsequently, absorbance was measured in the presence of NAP before and following irradiation at 385 nm, with 100% light intensity for 20 seconds.

[0776] For PB-17 a stock solution was prepared at a concentration of 0.1 mg / mL by dissolving 10 mg of PB-17 in 100.04 gram of 10 mM HC1, using an analytical balance and a 3 mL pipette. From this solution, 1 mL was transferred to a disposable cuvette for spectral measurement. A blank sample was prepared by transferring 1 mL of 10 mM HC1 to a separate cuvette. Absorbance spectra were recorded using spectrophotometer. To evaluate UV stability in the presence of NAP prior to irradiation, a 1 mL solution containing 0.1 mg / mL PB-17 and 0.2 mg / mL NAP in 10 mM HC1 was prepared. The absorbance spectrum was recorded. The solution was then irradiated at 385 nm with 100% intensity for 20 seconds and the absorbance spectrum was once again recorded.

[0777] For PB-23 a stock solution was prepared at a concentration of 1 mg / mL by dissolving 10 mg of PB-23 in 10.01 gram of absolute ethanol. This stock was diluted 1:40 by mixing 200 pL of the stock solution with ethanol to obtain a final concentration of 0.025 mg / mL. A 1 mL aliquot of the diluted solution was transferred to a disposable cuvette for measurement. A blank was prepared using 1 mL of absolute ethanol. The absorbance spectrum was measured using spectrophotometer. For UV stability testing in the presence of NAP prior to irradiation a 1 mL solution containing 0.025 mg / mL PB-23 and 0.2 mg / mL NAP in ethanol was prepared. After the absorbance spectrum was recorded, the solution was irradiated under the same conditions described previously and the absorbance spectrum was once again recorded.

[0778] For PB-22 a stock solution of 1 mg / mL was prepared by dissolving 10 mg of PB-22 in 10.02 gram of 10 mM HC1. The stock solution was diluted 1:40 to achieve a final concentration of 0.025 mg / mL. A 1 mL portion of the diluted solution was transferred to a disposable cuvette, and the blank consisted of 1 mL of lOmM HC1. The absorbance spectrum was recorded using spectrophotometer. To assess the stability of PB-22 under UV exposure, a 1 mL solution containing 0.025 mg / mL PB-22 and 0.02 mg / mL NAP was prepared. After the absorbance spectrum was recorded, the solution was irradiated under the same conditions described previously and the absorbance spectrum was once again recorded.

[0779] Method 4 Compatibility in formulation

[0780] Compatibility of tested photoblockers within different curable formulations was typically determined by visual inspection whereby lack of sedimentation was indicative of a homogeneous solution.

[0781] Method 5 Curing kinetics

[0782] The curing (e.g., as a result of polymerization and / or cross-linking) kinetics of different formulations were measured using Discovery HR-2, a stress-controlled rheometer, equipped with a 20 mm parallel plate geometry and an Omnicure (series 2000) optics attachment as the light source. A 20 mm diameter parallel plate spindle was paired with a quartz bottom plate to allow for in situ photo-irradiation. All experiments were performed at a controlled temperature (e.g., 22 °C). 25-90 pL (e.g. ,80-85 pL) of each sample were loaded onto the bottom plate (using a viscous liquid pipette to ensure uniform deposition) and the top geometry was lowered to obtain a gap size of 50- 250 pm (e.g., 250 pm), respective to the drop volume. Measurement’s duration was set to 120 seconds.

[0783] Each sample was conditioned pre-shear in 1.0 rad / s for 10 seconds to homogenize the sample structure prior to oscillatory shear testing. Then oscillation fast sampling test was initiated with angular frequency of 2 Hz and a 10 % strain, 30 seconds delay to reach a pre-irradiation equilibration. Subsequently samples were exposed to UV irradiation at a wavelength of 365 nm, for 60 seconds at intensity of 50 mW / cm2followed by an additional 30 sec measurement. Oscillatory shear was continuously applied during irradiation and for the additional 30 second post-irradiation.

[0784] The storage (G’) and loss (G”) moduli were continuously recorded throughout the experiment, and values were averaged (each formulation was tested for a minimum of 3 replicates) over the period of 100-120 seconds post-irradiation for analysis. The kinetic parameter for evaluation, which is associated with the steep increase in G' values in response to illumination, is time to reach G' >100 Pa. Shorter time to reach G' > 100 Pa reflects faster curing kinetics.

[0785] For example, to obtain good resolution for formulation F25 the time to reach G' >100 Pa was determined to be less than 11 sec as poor resolution was observed when Time to G’ > 100 exceeded 11 sec. As shown in Example 6 when Time to G’ > 100 was 3.78 sec good resolution was obtained in the xy and z dierections. TR delays the time required for the material to completely cure, meaning to reach the G’ plateau (14+ / -6 KPa), once exposed to the UV illumination (see Example 6).

[0786] For example, when time to reach G' >100 Pa was 1.32+ / -0.28sec for formulation H22 good resolution in the xy and z directions was observed. HC9 delays the time required for the material to completely cure, meaning to reach the G’ plateau (G’= 15.97+ / - 8.8 KPa), once exposed to the UV illumination (see Example 4).

[0787] In an alternative procedure, the curing kinetics of different formulations (e.g., formulation H5) were measured using same Discovery HR-2 rheometer and each sample was subjected to conditioning at 22 °C, pre-shear in 1.0 rad / sec for 10 seconds, oscillation fast sampling, 10 % strain at 2 Hz, 30 seconds delay, followed by 7.5 mW / cm2irradiation for 120 seconds, followed by additional 30 seconds measurement. G' values were averaged at 100-120 seconds, time to G' was set as the first time point in which G' was > 100 Pa.

[0788] Method 6 Printing resolution

[0789] Printing resolution refers to how accurately the final printed part replicates the original CAD (Computer-Aided Design) model. The printed structure should exhibit precise, well-defined features, closely matching the intended dimensions with minimal deviation. As full similarity is unachievable, an acceptable tolerance range must be defined to specify the permissible variation between the designed model and the actual printed part.

[0790] A combination of formulation properties and printing parameters should reach an optimum to achieve this property. Thus, the formulation kinetics and rheology properties should allow accurate printing, while the printing parameters should be optimized per formulation. The main properties or components of the formulation which may affect the resolution of the printed part are the photo-blocker and initiator reaction and concentration, the number of polymeric crosslinking sites, and the viscosity. Multiple printing parameters may affect the resolution; however, the main parameters are energy-dose, layer thickness, and platform movement.

[0791] The amount of energy delivered by the light source to every cured layer. The energy is set by the user and should be changed for every formulation. If the energy is too low, the formulation will not cure properly. If the energy is too high, the formulation will overcure. In both cases, the resolution will be damaged. The energy should be set correctly, depending on how reactive the formulation is, which is mainly affected by the photo blocker and initiator, and the number of sites available in the formulation.

[0792] In general, thinner printed layers result in smoother surfaces and higher accuracy. However, achieving this requires higher concentration of photoblocker to prevent light penetration into underlying layers, which significantly extends the overall printing time. To balance resolution with throughput - while minimizing print duration and maintaining acceptable resolution - the layer thickness was set to 150 microns, with photoblocker and initiator concentrations optimized accordingly.

[0793] The speed, length of travel, and other parameters related to the movement of the platform on which the part is printed may also affect the resolution. These parameters are mainly used to compensate for differences in viscosity between formulations. In general, the higher the viscosity of the formulation, the platform movement should be slower. The slower speed allows the liquid formulation to settle and reach equilibrium prior to the curing of the next layer.

[0794] To assess the resolution achieved with each formulation, a unique resolution model was developed. The features on this model were designed to present the required information to the user. The two models that were designed to assess resolution quality are the ‘XYZ’ and ‘United!’ models presented in FIG.6Aa-c and and FIG. 6Ad , respectively. The former provides a qualitative examination, while the latter shows a representation of the produced lattice-based scaffolds. Specifically, as the hydrogel cures under defined printing properties, including control on the energy doses applied to each layer, the printed features’ shape and sharpness indicate phenomena such as under- / over-polymerization. For example, a blurred shape of either the whole model or its features indicates under-polymerization, while closure of pores in Z-view suggests that overpolymerization was produced. Representative resolution test images of the considered formulation were captured and analyzed, showing sufficient quality resolution and accuracy in both XY and Z.

[0795] Resolution models were printed with B9-creations 6MPro, or with B9-creations 5XL 3D printers, with layer thickness ranging between 30 to 150 micrometers and energies ranging between 30mJ / cm2to 600mJ / cm2e.g., 120 mJ / cm2. An isometric view of the resolution model is presented in FIG. 6Aa, the XY plane resolution in FIG. 6Ab and the Z axis resolution in FIG. 6Ac. FIG. 6Ad presents a drawing of both views of a unit cell resolution model used to ensure sufficient resolution is kept in XY (left) and Z (right) while printing a representative part of the SC breast scaffold. Resolution models were printed to assess the printability of a formulation with sufficient resolution for the manufacturing of breast implants. Printing optimization process was conducted for both printing properties (e.g., energies) and formulation components’ properties (e.g., concentration).

[0796] Method 7 mechanical properties

[0797] All material specimens and lattice-based implants were 3D printed in the same DLP- technology-based printer. 3 tests were used for the evaluation of the mechanical properties: material tensile test using dogbone- shaped specimens, material compression test using cylindershaped specimens, and lattice compression test using 20cc breast scaffolds. See Figure 6L and 6M right panels for additional information on the dimensions of the specimens. Specimens and scaffolds were tested in a uniaxial compression test at a quasi-static displacement rate of 10 mm / min in a universal testing machine (Instron 68SC-1). Displacement and load were recorded during each test, and the displacements were post-processed to engineering strain based on the undeformed shape of the scaffolds. Since scaffolds vary in diameter along their ‘projection’ axis, the results are presented in a load-strain plot rather than the common stress-strain plots produced for the material tensile and compression specimens. Cylinders were placed on a small compression Instron’ s plate, while dogbones were installed on Instron’ s pressure-based grip while constantly being held by a pneumatic pressure of about 0.5 bar. For both material specimens, force is post processed to stress values by dividing it with the undeformed cross section area of the specimens.

[0798] Method 8 Cell Viability

[0799] In an exemplary procedure (H7 Example 4), disc-shaped models made of a curable formulation that comprises CMR and PEG-based polymerizable materials were placed in a 6-well plate (7 models per well) and irradiated with 254 nm UV lamp for 15 minutes. Models were submerged in 5 ml 70 % Ethanol solution for 30 minutes, washed, submerged in DPBS for 20 minutes, washed again and then kept in DPBS until cell seeding.

[0800] NHDF cells solution was diluted 1: 1 with 30 pl of Trypan blue. Cells were counted using Countess II automated cell counter. A total of 20K cells was placed on top of each mini-mesh. 50 pl of cell suspension with a total of 50K cells was placed on top of each Disc. Cells were incubated on models for 10 minutes, and then were topped with 700 pl of DMEM medium. Plates were incubated at 37 °C, 5 % CO2.

[0801] Following 5, 8 and 15 days of incubation, medium was aspirated from calibration curve plate wells and meshes. Prepared working solution of 1: 10 Presto Blue in DMEM medium was added to each well and the plates were incubated at 37 °C + 5% CO2 for 1.5 hours. 2x100 pL from each of the wells were transferred into a 96-wells plates. The fluorescence was read in the plate reader - excitation 560 nm, emission 590 nm.

[0802] Cell proliferation assay on meshes printed from H22 (Example 4) Primary Normal Human Dermal Fibroblasts (nHDF; PromoCell) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sartorius) supplemented with 10% (v / v) fetal bovine serum (FBS; Capricorn) and 100 pg / mL Primocin, at 37°C in a humidified atmosphere containing 5% CO2. Prior to cell seeding, the printed mesh constructs (see FIG. 60) were sterilized by incubation in 70% ethanol and then placed into the wells of a 24-well cell culture plate. Cells were then seeded directly onto the surface of each mesh at a density of approximately 20,000 or 50,000 cells per mesh. The seeded meshes were incubated under standard culture conditions (37°C, 5% CO2) for 11 days. Cell proliferation on the meshes was evaluated at multiple time points using the resazurin-based Presto Blue assay (Invitrogen). At each time point, a calibration curve was prepared and samples were incubated with the assay reagent for 2 hours before fluorescence measurement. As a control, cells were also seeded directly onto the tissue culture plastic of the well plate under identical conditions to confirm cell viability and proliferation capacity).

[0803] Fluorescence imaging on meshes printed from H22 (Example 4).

[0804] After 11 days of culture on the printed meshes, cells were fixed with 4% paraformaldehyde (PFA) and subsequently permeabilized using Dulbecco’s Phosphate-Buffered Saline (DPBS) containing 0.1% Triton X-100. Following fixation and permeabilization, cells were stained with NucBlue for nuclear visualization, and either Actin Green or Actin Red to label the actin cytoskeleton. Fluorescent images were acquired using an EVOS fluorescence microscope.

[0805] Method 9 Chemical Analytics

[0806] Chemical Stability of PB-13, PB-22, PB-23 Post Irradiation at 240J -HPLC (Example 9)

[0807] A solution of 0.44mg / mL PB-13 in 0.01M HC1, 0.15 mg / ml of PB-22 and 0.106 mg / ml PB23 each in O.lmL acetic acid were prepared. In order to confirm system suitability, standard solution was injected as control. Each PB solution was irradiated for 5 min at 385nmx 100% energy (corresponding to about 240 J equivalent to 8OOmW*3OO sec) in UV oven (UVATA). 0.1 ml of solution was diluted with 0.9 mL of mobile phase and injected. Non-irradiated solutions were similarly prepared and injected for comparison. The cured and non-cured samples were tested using Arc HPLC (Waters) using the following protocol:

[0808] Injection volume: 10 uL

[0809] Sample temperature =25 °C

[0810] Column temperature = 25 °C

[0811] Column BEH C18 3.5um 4.6x150mm

[0812] Flow rate= ImL / min

[0813] Detector: PDA 2998, wavelength = 365 nm.

[0814] Mobile phase A: 1 mL of formic acid & 900 mL of DDW+ 100 mL of Acetonitrile. Mobile phase B: 1 mL of formic acid&lOO mL of DDW+ 900 mL of Acetonitrile.

[0815] Needle wash solution: mobile phase A.

[0816] Dilution solution: mobile phase A.

[0817] Method 10 Accelerated and Real Time In-Vitro Degradation

[0818] For both studies 100 mL of each formulation F23 or F25 were prepared and 3.5cc implants scaffold depicted in Figure 24D (SC square pores) samples were printed using printing parameters of 140mJ / 400mJ; 50 pm of B9. 3 replicates x 4 timepoints were used for accelerated study and 5 replicates x 4 timepoints used printed for real time study.

[0819] Immediately after printing, the samples were transferred into a washing container and washed thoroughly with 200 ml of DDW using a washing bottle until all the residual non curable formulation was washed out. Samples were then post-cured in oven (385 nm; 40% intensity for 10 seconds) and subsequently washed with DDW for at least 30 minutes, repeated three times until the solution ran clear. Samples were frozen at -80°C and lyophilized overnight. Each dried implant was weighed using an analytic balance and the TO weight and corresponding weights and identification numbers were recorded for TO.

[0820] For both studies, each implant was incubated in 8 mL of degradation media, ensuring a minimum ratio of 1 g sample per 10 mL media, at 37°C in a shaker incubator. The media used for real time degradation study was Phosphate Buffered Saline (PBS X 1) pH 7. Relevant samples were removed from degradation solutions at each termination time point (3, 6, 9, 12 months) and placed on a well-labelled plate.

[0821] The media used for accelerated degradation study was PBS X 1 pH 7 for control samples, bicarbonate / carbonate buffer preparation pH 10 or 6% Hydrogen Peroxide (dilution -of 30% Hydrogen Peroxide solution) pH 6. Relevant samples were removed from degradation solutions at each termination time point (2, 7, 14 days or until dissolved) and placed on a well-labelled plate.

[0822] The samples were rinsed with DDW then refrozen at -80°C and lyophilized overnight. Mass loss of 3.5CC implants was calculated using the following equation % Mass loss= (W0- Wd) / Wd *100% and recorded and where Wo is the initial dry mass and Wd is the degraded dry mass. Visual inspection was also conducted at each termination time.

[0823] Method 11 Swelling

[0824] Cylinder and dogbone samples were post-cured ((385nm, 100% intensity for 20 seconds in custom made oven). Samples were weighed upon post cure and following immersion for 24h in Double Distilled Water (DDW) conducted prior to the mechanical testing. The swelling ratio was calculated as follows: (Weight after swelling-weight before printing) / (weight before printing) *100. Method 12 Viscosity

[0825] Viscosity measurements were performed using a cone-plate geometry on a stress- controlled rheometer. The spindle consisted of a 40 mm diameter, 10angle cone fabricated from ultra-high purity (UHP) stainless steel. The bottom plate was planar and temperature controlled. A 300 pL volume of each sample was dispensed onto the rheometer plate using a viscous liquid pipette to ensure accurate placement and minimize air bubble inclusion. Samples were conditioned at 22 °C with a 10-second soak time to allow thermal equilibration prior to mechanical testing. A pre-shear protocol of 2.0 rad / s for 20 seconds was applied to ensure structural uniformity and reproducibility between replicates.

[0826] Viscosity measurements were performed using the pre-defined rheometer protocol. The method consisted of a logarithmic flow sweep from a shear rate of 0.1 to 1000 s ', with three data points collected per decade. Steady-state sensing was employed for each data point, with a maximum equilibrium time of 60 seconds, a sampling period of 5 seconds, and a convergence tolerance of 5%. Each formulation was tested for a minimum of 3 replicates (unless indicated differently), and viscosity values were collected and averaged at 10 1 / sec.

[0827] Mechanical drawings

[0828] In the mechanical drawings each figure is divided into four subfigures. The main subfigure appearing at the top panel, presents the stress-strain (or load-strain) curves. A legend indicating the line type and (grayscale) color is available on the top-left side of each subfigure. The bottom panel of the figure presents bar-plots of three main mechanical properties derived from the main subfigure. The strength (or load-limit), strain-at-break, and accumulated energy subfigures appear on the left-, middle-, and right-hand side of the bottom panel respectively. Within each of these subfigures, bars are ordered from left to right correlating to the main subfigure order from top to bottom (e.g., the top group in the legend of the main subfigure will appear as the left bar in each subfigure), and are filled with a pattern colored in the same color as the lines in the main subfigure. Tensile mechanical drawings of dogbone specimens present only the top half of the specimens ranked by their performance. The number of dogbone specimens tested varied between 10-15, and out of the valid tests, only half were selected for generation of the stress-strain curves and bar plots. The reason for such selection was to better distinguish between different groups (e.g., different formulations). The bottom half of the specimens were consistent in stiffness compared to the rest, yet at times, they were weaker due to local stress concentrations. Therefore, as the comparison between formulations was based on the top half of the tensile specimens, it was effectively compared to the tensile mechanical potential of the formulation. In the compression tests, all cylinders and scaffolds were selected for plot generation as their mechanical properties were more consistent inter alia due to simpler testing fixtures) thus fewer specimens were printed for testing (about 5 cylinders and up to 3 scaffolds).

[0829] EXAMPLE 3

[0830] Results for exemplary PBs

[0831] Dye substance candidates were compared to known photoblockers, minocycline (PB-1) and Quinoline Yellow (PB-2).

[0832] The following dye substances lacked sufficient solubility in an aqueous carrier (e.g., DDW or 10 mM HC1) or UV-absorbance: PB-18, PB-19, PB-20, PB-21, PB-16 and PB-10. Although PB- 6 was soluble, it underwent rapid degradation and lost its absorbance.

[0833] PB-3, PB-4 and PB-10 demonstrated lack of absorbance at the selected wavelength (385 nm) at high concentrations (ImM in lOmM HC1 solutions) (not shown), as opposed to lower concentration O.lmM, (FIG. 1A). ImM in lOmM HC1 solutions of PB-7 and PB-9 (FIG. IB) demonstrated absorbance at the selected wavelength similar to ImM in lOmM HC1 solutions of PB-1 and PB-2 prior and post irradiation (365 nm 100% 20sec).

[0834] However, when absorbance was measured for 1 mM PB in lOmM HC1 solutions that further comprise a photoinitiator (e.g., 0.5% NAP), it was surprisingly uncovered that some of the tested substances (PB-1, PB-2, PB-5, PB-7, PB-8, PB-9, PB-11, PB-12, PB-13, PB-14, PB-15, PB-16) changed their color and / or lost their absorbance following irradiation (365nm 100% 20sec), as shown in FIGs. 2A and 2B respectively. In contrast PB-13 and PB-14 in the presence of NAP maintained absorbance after irradiation (365nm 100% 20sec), as shown in FIG. 2C in addition to the PB-1 and PB-2 (which served as positive controls), as shown in FIG. 2B.

[0835] FIG. 3A shows that 0.27 mM PB-19 exhibited no absorbance at the wavelength range of interest (bottom curve), whereas 0.2 mM PB-17 did.

[0836] PB-17 (0.2 mM), was stable following irradiation (385 nm 100% 20sec) in the presence of NAP, as presented in FIG. 3B. As absorbance values were not included in the linear range per Beer Lambert equation, the samples were re-prepared at a lower concentration (PB17 O.lmg / mL) and photoabsorbance was reevaluated and confirmed as stable prior and post irradiation (not shown).

[0837] Printing resolution measurements were then performed for PB-17. The energy dose was primarily set at 120 mJ / cm2, yet resulted in over-polymerization at XY and Z. The energy dose was therefore reduced to 60 mJ / cm2and then to 30 mJ / cm2. FIGs. 4A-B present photographs of a resolution model demonstrating the resolution obtained for a formulation comprising 0.004 % by weight PB-17, 0.5 % by weight of CMR, 15%PEG-based polymerizable materials (PEGDA 3.4K), and 0.5 % NAP.

[0838] As shown therein, at 30 mJ / cm2, sufficient polymerization at XY (FIG. 4A) and low resolution at Z (FIG. 4B) due to over polymerization in Z were observed.

[0839] EXAMPLE 4 - PB-13

[0840] In DLP printing the bioink needs to be formulated with a photoinitiator and a photoblocker to enable printing with high resolution on the XY and Z direction. PB-13 was selected for further analysis with photocurable biological material with or without polymer.

[0841] Various formulations comprising PB-13 were prepared to test kinetics, printability, mechanical and biological properties. The formulations are described in Tables 2-4 herein above:

[0842] RESULTS

[0843] FIG.5 presents the data obtained in kinetic studies using Formulation H5, indicating that PB-13 at a concentration of 0.04 % by weight did not affect the storage modulus of the cured formulation and did not increase the curing rate.

[0844] Printing resolution of H4 printed at an energy of 35mJ / cm2, using a calibration model as shown in FIG. 6Aa-6Ac confirmed this dye substance to be a suitable candidate with respect to resolution, as shown in FIG. 6B compared with the same formulation with MC. Formulations H2 and H3 both printed at an energy of 35mJ / cm2were found to have better resolution than Hl and H4 (data not shown). H8 and H9 printed at an energy of 80mJ / cm2and 90mJ / cm2respectively resulted in the highest printing resolution in both XY plane and Z direction (FIG. 6C and 6D respectively). While the resolution of H4 may be acceptable for applications requiring lower printing resolution, increasing the concentration of HC9 from 0.02% to 0.04% as demonstrated for H8 or H9 may improve the resolution of H4 for applications requiring higher resolution.

[0845] FIGs. 7A-D present the mechanical properties for models printed from a formulation that comprises 0.08% PB-13 (H6 printed at 5mWcm'2), compared to the same formulation comprising 0.5%MC (F0 as shown in Table 9 printed at 27mWcm’2). These models were tested according to Method 7, showing that the specimens featured the desired mechanical properties; i.e., dogbones printed with PB-13 reached higher strain at break and strength average values than those printed with MC, whilst maintaining the approximate tensile stiffness.

[0846] FIG. 8 presents the data obtained for cell quantification by presto blue proliferation assay, of models made of formulations H7 that comprise 0.04% PB-13, or the same formulation (F01 as shown in Table 9) with 0.2% MC), at day 5, 8 and 15, in accordance with Method 8 described in Example 2. This data demonstrates successful identification of PB-13 as a photoblocker that is suitable for inclusion in curable formulations comprising CMR and photocurable polymers that are usable in 3D-bioprinting.

[0847] PB-13 was selected for further analysis for printability with different photocurable biological materials other than CMR. Various formulations comprising an aqueous carrier, GelMA or COLLMA, a photoinitiator, PB-13 and optionally at least one polymer as set out in Table 5 were prepared and tested for printability.

[0848] Formulation H10 (corresponding to H3 but with 0.5%GelMA in PBS instead of 0.5%CMR in lOmM HCL) was slightly turbid upon preparation and post overnight storage at 4 °C than a comparable formulation with CMR. Unlike H3, a resolution model printed from H10 at the same energy (35mJ / cm2) resulted in poor resolution in both XY and Z directions. Increasing energy to 120mJ / cm2, improved the resolution in the XY direction, but resulted in over curing in the Z direction (FIG. 6E& F respectively). Formulation H13 corresponding to H10 where the solvent used to dissolve GelMA was replaced (lOmM HCL instead of PBS) resulted in a clear formulation upon preparation and post overnight storage at 4°C. Increasing PB 13 concentration from 0.02% to 0.03%, resulted in improved resolution in all directions and the highest resolution was achieved when printed at energy 110mJ / cm2(FIG. 6G). In addition, the texture of the hydrogel was smoother (less grainy) than that of H10.

[0849] When GelMA was replaced with CollMA, in formulation H12 (corresponding to Hl 3) it was not well dissolved and gel fragments were observed upon preparation. Additional mixing was performed on the next day on roller mixer for 1 hour post overnight storage at 4°C prior to printing at an energy of 200mJ / cm2. Although the resolution of H12 in XY&Z directions was deemed acceptable (FIG. 6H), it was not as good as CMR / GelMA-based formulations.

[0850] Assuming the methacrylate level of CollMA is similar to CMR50 it may be possible to improve resolution by increasing the concentration of PB-13. Thus, HC9 was found to be a suitable PB for use with curable biological material when combined with 15%PEGDA.

[0851] Hl 1 comprising GelMA dissolved in PBS and free of polymers was clear upon preparation post overnight storage at 4°C. Hl l required high energy dose for polymerization and even when printed at an energy of 400mJ / cm2resulted in unacceptable resolution at XY, (FIG. 61). Thus, a higher concentration of GelMA or the presence of polymers in such formulation is deemed critical to allow printability. Without being bound by any theory, lack of homogeneity of H10 as opposed to Hl 1 may be attributed to the polymerization reaction between PEGDA and GelMA resulting in sedimentation. Unlike H10, Hl l does not have polymers required for printability resulting in a poor resolution in XY direction. HC9 CMR and no polymers

[0852] In DLP (Digital Light Processing) printing, the bioink must be formulated with specific printing aids - namely a photoinitiator and a photoblocker — to achieve high-resolution printing in both the XY plane and the Z direction. The goal was to develop a new bioink for DLP bioprinting which is free of polymers composed of methacrylated rhCollagen, a photoinitator, and a photoblocker. PB-13 was selected as a photoblocker for further analysis. Biological, mechanical and printability properties of the printed constructs were defined, and formulations were developed and tested accordingly.

[0853] Various formulations comprising an aqueous carrier, rhCollagen varying in degree of methacrylation (50% or 90% also referred to herein as CMR50 or CMR90 respectively), a photoinitiator (LAP for all formulations other than H15 which had NAP) and photoblocker (PB- 13) were prepared as set out in Table 6. Resolution models (calibration or UC models) of each of the tested formulations were printed at different energies and concentrations of each formulation component were adjusted to reach the optimal resolution in the XY plane and Z direction (not shown). Printing conditions were developed and optimized for 6MPro B9C DLP.

[0854] The highest printing resolution models having a layer thickness of 150 pm were achieved for formulations comprising either highly methacrylated collagen (CMR90) (FIG. 6 J left H15 220 mJ / cm2 settle time range between 0-40 seconds) or a high concentration of Collagen-MA (20mg / ml CMR-50L) (FIG. 6J right H22 160-200mJ / cm2 settle time range between 120-200 seconds). The printing resolution in XY plane for H15 enabled printing 1mm X 1mm sharp and clear holes. In addition, the thick bold digits are clear and readable. In Z direction, the 1.5mmX1.5mm and larger holes were well printed while the Immxlmm was closed. A longer settle time provides the necessary time for the formulation to stop flowing. As H22 was found to be more viscous than H15 a longer settle time of the printing platform at the formulation level was therefore selected to improve the printing resolution. The printing resolution of H22 in XY plane enabled printing ImmXlmm sharp and clear open holes. In addition, the thick, bold digits were clear and readable. In Z direction, the...

Claims

WHAT IS CLAIMED IS:

1. A photoblocker usable in additive manufacturing a three-dimensional object, being represented by Formula I:Re, R7, Rs, R9 and Rio are each independently hydrogen; andRi, R2, R3, R4 and R5 are each independently selected from hydrogen, hydroxyalkyl and a saccharide moiety.

2. The photoblocker of claim 1, wherein said saccharide moiety is selected from a monosaccharide and disaccharide moiety.

3. The photoblocker of claim 1 or 2, wherein said saccharide moiety comprises a glucose moiety.

4. The photoblocker of any one of claims 1 to 3, wherein R3 is said saccharide moiety.

5. The photoblocker of any one of claims 1 to 4, wherein Ri is hydrogen.

6. The photoblocker of any one of claims 1 to 5, wherein R2, R4 and Rs are each hydrogen.

7. The photoblocker of any one of claims 1 to 5, wherein R2, R4 and Rs are each a hydroxyalkyl.

8. The photoblocker of any one of claims 1 to 7, wherein said hydroxyalkyl is hydroxyethyl.

9. The photoblocker of claim 1, wherein R3 is said saccharide moiety; and Ri, R2, R4 and R5 are each hydrogen.

10. The photoblocker of claim 9, wherein said saccharide moiety is rutinose, the photoblocker being 3-glucoside quercetin.

11. The photoblocker of claim 1, wherein R3 is said saccharide moiety; Ri hydrogen; and R2, R4 and R5 are each independently a hydroxyalkyl.

12. The photoblocker of claim 11, wherein each of R2, R4 and Rs is hydroxyethyl.

13. The photoblocker of claim 12, wherein said saccharide moiety is rutinose, the photoblocker being troxerutin.

14. A photoblocker usable in additive manufacturing a three-dimensional object, the photoblocker being represented by formula I*Formula I* whereinRx and Ry are each independently selected from hydrogen (H), alkyl (e.g., lower alkyl as described herein, for example, of 1 to 4 carbon atoms), cycloalkyl and aryl; and Rz is an alkyl (e.g., lower alkyl as described herein, for example, of 1 to 4 carbon atoms), aryl or cycloalkyl.

15. The photoblocker of claim 14, being HC yellow 9 (HC9).

16. The photoblocker of any one of claims 1-15, characterized by at least one of:biocompatibility and non-toxicity at formulation concentration prior and post curing; solubility of at least 0.05 mg / mL in an aqueous carrier or a curable formulation; absorbance at wavelength from 300 nm to 800 nm; absorbance stability at said wavelength in the presence of a photoinitiator; or chemical stability post irradiation at 240J.

17. A curable formulation usable in additive manufacturing a three-dimensional object featuring, in at least a portion thereof, a biological material, the formulation comprising a photoinitiator, a photocurable biological material, a carrier and the photoblocker according to any one of claims 1 to 16.

18. The curable formulation of claim 17, having components as set forth in any one of Tables A-Z, AA or AB.

19. The curable formulation of claim 17, having components as set forth in any one of Tables J-U.

20. The curable formulation of claim 17, having components as set forth in Table K or Table R.

21. The curable formulation of claim 17, wherein an amount of said photoinitiator in said curable formulation ranges from about 0.1 to 1 % by weight of the total weight of the formulation.

22. The curable formulation of claims 17 or 21, wherein said photoinitiator is an acyl phosphine oxide type photoinitiator.

23. The curable formulation of claim 22, wherein said acyl phosphine oxide type photoinitiator is 2,4,6-trimethylbenzolydiphenyl phosphine oxide (TMPO) or a salt thereof.

24. The curable formulation of claim 22, wherein said salt is lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) or sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP).

25. The curable formulation of any one of claims 17-24, wherein an amount of said photoblocker in said curable formulation ranges from about 0.02-0.5 % by weight of the total weight of the formulation.

26. The curable formulation of any one of claims 17-24, wherein said photocurable biological material comprises a collagen that features a plurality of photocurable groups.

27. The curable formulation of claim 26, wherein said photocurable groups comprise (meth)acrylic groups.

28. The curable formulation of claims 26 or 27, wherein the collagen is a human Type I collagen.

29. The curable formulation of any one of claims 26-28, wherein the collagen is a recombinant collagen.

30. The curable formulation of claim 29, wherein the collagen is a plant-derived recombinant collagen.

31. The curable formulation of any one of claims 26 to 30, wherein the collagen is a plant-derived recombinant human Type I collagen.

32. The curable formulation of any one of claims 26-31, wherein an amount of said collagen in said curable formulation ranges from about 0.1-2 % by weight of the total weight of the formulation.

33. The curable formulation of any one of claims 17-32, further comprising at least one synthetic biocompatible curable material that features a plurality of photocurable groups.

34. The curable formulation of any one of claims 17-32, further comprising a photocurable polymeric material.

35. The curable formulation of claim 34, comprising components as set forth in any one of Tables B-D, F, V and Z.

36. The curable formulation of any one of claims 17-32, further comprising at least two photocurable polymeric materials.

37. The curable formulation of claim 34 or 36, wherein an amount of said collagen in said curable formulation ranges from 0.1-1 % by weight of the total weight of the formulation.

38. The curable formulation of claim 36, wherein: at least one of said at least two photocurable polymeric materials has an average molecular weight lower than 10,000 grams / mol (low MW photocurable polymeric material), and at least another one of said at least two photocurable polymeric materials has an average molecular weight higher than 10,000, or higher than 15,000 (high MW photocurable polymeric material), grams / mol; a total amount of said at least two photocurable polymeric materials is in a range of from 1 to 20, or from 5 to 20, or from 5 to 15, or from 7.5 to 15, or from 7.5 to 10, % by weight, of the total weight of the curable formulation; and a weight ratio between said low MW photocurable polymeric material and said high MW photocurable polymeric material ranges from 1: 1 to 1:9, or from 1: 1 to 1:5.

39. The curable formulation of claim 38, wherein each of said photocurable polymeric material independently is a multifunctional photocurable polymeric material featuring two or more photocurable groups.

40. The curable formulation of claim 39, wherein said photocurable groups are (meth)acrylic groups.

41. The curable formulation of claim 40, wherein said (meth)acrylic groups are selected from acrylate and methacrylate groups, provided that at least one of said photocurable polymeric materials features acrylate groups.

42. The curable formulation of claim 41, wherein one of said at least two photocurable polymeric materials features a plurality of acrylate groups and another one of said photocurable polymeric materials features a plurality of methacrylate groups.

43. The curable formulation of any one of claims 38 to 42, wherein at least one of said photocurable polymeric materials comprises a poly(alkylene glycol) polymeric backbone.

44. The curable formulation of any one of claims 38 to 43, wherein said low MW photocurable polymeric material is a poly(alkylene glycol) that terminates by two (or more) acrylate or methacrylate groups.

45. The curable formulation of any one of claims 38 to 44, wherein an average molecular weight of said low MW photocurable polymeric material ranges from 500 to 10,000, or from 500 to 5,000, or from 500 to 4,000, or from 500 to 3,500, or from 1,000 to 10,000, or from 2,000 to 10,000, or from 1,000 to 8,000, or from 2,000 to 8,000, or from 1,000 to 6,000, or from 2,000 to 6,000, or from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 6,000 or from 3,000 to 5,000 or from 3,000 to 4,000, or from 1,000 to 3,500, or from 500 to 3,500, or from 1,000 to 3,000, or from 1,000 to 2,000, grams / mol.

46. The curable formulation of any one of claims 38 to 44, wherein said high MW photocurable polymeric material comprises a poly(alkylene glycol) polymeric backbone and terminates by two (or more) acrylate or methacrylate groups.

47. The curable formulation of claim 46, wherein an average molecular weight of said poly (alkylene glycol) polymeric backbone is higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol.

48. The curable formulation of claim 46 or 47, wherein an average molecular weight of said poly (alkylene glycol) polymeric backbone ranges from 10,000 to 40,000, or from 10,000 to 30,000, or from 15,000 to 40,000, or from 15,000 to 30,000, or from 20,000 to 40,000, or from 20,000 to 30,000, or from 20,000 to 25,000, grams / mol.

49. The curable formulation of any one of claims 46 to 48, wherein said high MW photocurable polymeric material is a multi-block (e.g., diblock or triblock) co-polymer that comprises said poly(alkylene glycol) polymeric backbone as one block and at least one additional block that comprises a polymeric backbone of a biodegradable polymer other than poly(alkylene glycol).

50. The curable formulation of claim 49, wherein said high MW photocurable polymeric material is a tri-block co-polymer that comprises said poly(alkylene glycol) polymeric backbone as a middle block having attached to its termini two additional blocks, each comprising a polymeric backbone of a biodegradable polymer other than poly(alkylene glycol) that terminates by a photocurable group.

51. The curable formulation of claim 49 or 50, wherein at least one additional termini block of said biodegradable polymer is a polyester.

52. The curable formulation of any one of claims 49 to 51, wherein at least one additional termini block of said biodegradable polymer is poly(caprolactones) (PCL).

53. The curable formulation of any one of claims 49 to 52, wherein a molecular weight of said at least one additional termini block is lower than 5,000, or lower than 3,000, or lower than 2,000 grams / mol.

54. The curable formulation of any one of claims 49 to 53, wherein a molecular weight of said at least one additional termini block ranges from 500 to 5,000, or from, 500 to 4,000, or from 500 to 3,000, or from 500 to 2,000, or from 1,000 to 5,000, or from 1,000 to 4,000, or from 1,000 to 3,000, or from 1,000 to 2,000, or from 1,400 to 1,600 grams / mol.

55. The curable formulation of any one of claims 38 to 54, wherein said high MW photocurable polymeric material is a triblock polymeric material that comprises a poly(alkylene glycol) polymeric backbone having a MW higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol and having attached to each of its termini a block of a polyester having a MW lower than 5,000, or lower than 3,000, or lower than 2,000 grams / mol and terminating by at least one (meth) acrylate group.

56. The curable formulation of claim 41, wherein: said low MW photocurable polymeric material is a poly(alkylene glycol) that terminates by two of said acrylate or methacrylate groups and has an average molecular weight that ranges from 1,000 to 10,000, or from 2,000 to 10,000, or from 1,000 to 8,000, or from 2,000 to 8,000, or from 1,000 to 6,000, from 1,000 to 5,000, from 1,000 to 4,000, from 1,000 to 3,000, from 1,000to 2,000, or from 2,000 to 6,000, or from 1,000 to 5,000 or from 2,000 to 5,000, or from 3,000 to 6,000 or from 3,000 to 5,000 or from 3,000 to 4,000 grams / mol; and said high MW photocurable material is a triblock polymeric material that comprises a poly(alkylene glycol) polymeric backbone having a MW higher than 10,000, or higher than 15,000 or higher than 20,000 grams / mol and having attached to each of its termini a block of a polyester having a MW lower than 5,000, or lower than 3,000, or lower than 2,000, or lower than 1,600 grams / mol and terminating by said (meth) acrylate group.

57. The curable formulation of claim 56, wherein said low MW photocurable polymeric material terminate by two acrylate groups.

58. The curable formulation of claim 56 or 57, wherein said high MW photocurable polymeric material terminates by two methacrylate groups.

59. The curable formulation of any one of claims 38 to 58, wherein a weight ratio between said low MW photocurable polymeric material and said high MW photocurable polymeric material is about 1:3.

60. The curable formulation of any one of claims 38-59, comprising components as set forth in any one of Tables E, G, H, I, W, X, Y, AA and AB.

61. The curable formulation of any one of claims 17-59, featuring a viscosity at 22 °C that ranges from 100 to 2,000 mPa- second.

62. The curable formulation of any one of claims 17-59, featuring a viscosity at 22 °C that ranges from 80-3000 centipoises (cP).

63. The curable formulation of any one of claims 17-59, featuring a viscosity at 22 °C that ranges from 70 to 2,500 mPa- second.

64. The curable formulation of any one of claims 17-59, featuring a viscosity at 22 °C that ranges from 200 to 2,500 mPa- second.

65. The curable formulation of any one of claims 17-59, featuring, when hardened a mechanical and / or rheological characteristic as set forth in Table 10.

66. The curable formulation of any one of claims 17-59, featuring, when hardened a tensile strain-at-break of at least about 100% and / or is capable of withstanding a tensile stress of IMPa.

67. The curable formulation of any one of claims 17-64, providing, when hardened, a biological or a biocompatible material featuring mechanical properties that meet the requirements of a breast implant.

68. The curable formulation of any one of claims 17-67, providing, when hardened, a biological or a biocompatible material featuring at least one of: strain of at least 20 % at 10-100 N; strain of at least 50 % at 100-200 N; compressive strain-at-break higher than 50%; compressive force limit of at least 156N; and volume retention under physiological stress of no more than 30 % strain, at 10N force, as determined in a in a compression force versus strain measurements as described herein.

69. The curable material of any one of claims 17-68, wherein said carrier is an aqueous carrier.

70. A process of additive manufacturing a three-dimensional object featuring, in at least a portion thereof, a biological or a biocompatible material, the process comprising sequentially exposing the curable formulation of any one of claims 17-68, to irradiation at a wavelength of from 300 to 800 nm, in a layerwise manner, and in a configured pattern corresponding to a shape of the object, thereby manufacturing the three-dimensional object.

71. The process of claim 70, wherein said exposing is for time period that ranges from 1 second to 120 or from 1 second to 20 second or from 1 second to 10 second for each layer or is about 12 seconds.

72. The process of claim 70 or 71 , wherein said irradiation is at a wavelength that ranges from 300 to 600 nm, or from 300 to 500 nm, or from 350 to 450 nm, or is 385 nm.

73. The process of any one of claims 70 to 72, wherein said irradiation is at a power intensity level that ranges from 1 to 150 mW / cm2or from 1 to 50 mW / cm2or is about 10mW / cm2.

74. The process of any one of claims 70 to 72, wherein said irradiation is at an energy level that ranges from 1 to 250 mJ / cm2or from 1 to 210 mJ / cm2or from 1 to 130mJ / cm2or is about 120mJ / cm2.

75. The process of any one of claims 70 to 74, wherein the additive manufacturing is DLP.

76. A kit comprising the photoblocker of any one of claims 1-16, a photoinitiator and a photocurable biological material.

77. The kit of claim 76, comprising components as set forth in Table K and Table R.

78. The kit of claim 76, further comprising a polymer.

79. The kit of any one of claims 76 to 78, wherein said photoinitiator is an acyl phosphine oxide type photoinitiator.

80. The kit of any one of claims 76 to 78, wherein said acyl phosphine oxide type photoinitiator is 2,4,6-trimethylbenzolydiphenyl phosphine oxide (TMPO) or a salt thereof.

81. The kit of any one of claims 76 to 78, wherein said salt is lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) or sodium phenyl-2,4,6-trimethylbenzoylphosphinate (NAP).

82. The kit of any one of claims 76 to 78, wherein said photocurable biological material comprises a collagen that features a plurality of photocurable groups.

83. The kit of claim 82, wherein said collagen is lyophilized.

84. The kit of any one of claims 76 to 78, wherein said photocurable groups comprise (meth)acrylic groups.

85. The kit of claim 82, wherein said collagen is wherein the collagen is a plant-derived recombinant human Type I collagen.

86. The kit of any one of claims 76 to 78, wherein the degree of collagen methacrylation is at least 37 %.

87. The kit of any one of claims 76 to 78, wherein the degree of collagen methacrylation is at least 50 %.

88. The kit of any one of claims 76 to 78, wherein the degree of collagen methacrylation is at least 90 %.

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