Collagen-based formulations that can be used as soft tissue fillers and / or implants - Patent Application 20070122999

Abstract

A three-dimensional (3D), biocompatible, degradable soft tissue implant comprising a bioprinted composite scaffold, the composite scaffold comprising recombinant human collagen (rhcollagen) and a biocompatible synthetic polymer, characterized by a porous wall, an internal cavity at least partially enclosed within the porous wall, and at least one injection port connecting the internal cavity to the outermost surface of the scaffold, the injection port having an opening sized to allow insertion of an injection device through the port. A soft tissue implant, a process for preparing the soft tissue implant, and use of the soft tissue implant in soft tissue reconstruction are also provided. An injectable matrix for use alone or in combination with an implant for soft tissue reconstruction is also provided.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 992,998, filed March 22, 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Statement The 49,152-byte ASCII file "86700 Sequence Listing.txt," created on March 22, 2021, which was submitted concurrently with the filing of this application, is hereby incorporated by reference as part of this specification.

[0003] Technical Field In some embodiments, the present invention relates to the repair and / or augmentation of soft tissue, and more particularly, but not exclusively, to collagen-based formulations that can be used to form degradable scaffolds and soft tissue implants comprising same, and collagen-based formulations that can be used as soft tissue fillers and / or in combination with degradable scaffolds. [Background technology]

[0004] Common soft tissue implants are used, for example, in the field of plastic surgery after mastectomy or in cosmetic breast augmentation surgery, in the form of breast implants. Further applications of soft tissue implants include, for example, calf muscle prostheses and cheek, nose, gluteal, testicular or upper arm muscle implants. Other examples of opportunities for soft tissue augmentation include the face, buttocks, hollow scar contours, or other body deformities or areas where augmentation is desired.

[0005] High requirements are placed on materials introduced into the human body, such as good biocompatibility. Particularly high requirements are placed on materials that are intended to remain in the body (permanently or temporarily) as implants. Medical implants have the function of supporting or replacing bodily functions, and in the case of plastic implants, restoring or changing the shape of body parts that may be destroyed.

[0006] Prosthetic implants have been developed for subcutaneous insertion. However, the severity of potential complications, such as scarring, implant rupture, capsular contracture, necrosis, and implant migration, as well as their recent negative publicity, have significantly reduced the desirability of such implants. Although scientific evidence is inconclusive, there are indications that breast implants may result in secondary morbidity. Therefore, there is a societal need for alternative means of accessing breast augmentation surgery.

[0007] Mammoplasty (cosmetic breast surgery) involves breast augmentation to enhance breast size, shape, and feel, or reconstruction after damage to breast tissue due to trauma, disease (breast cancer), and anatomical deformity. In the United States, breast augmentation has been the most commonly performed cosmetic surgical procedure since 2006 (American Society of Plastic Surgeons, 2018, Plastic Surgery Statistics Report). The main types of breast implants used are saline-filled implants and silicone gel-filled implants. The shells of both types of implants are manufactured from polysiloxane silicone rubber. Complications after breast augmentation include breast pain, changes in sensation, impaired lactation, visible wrinkling, asymmetry, and thinning of breast tissue.

[0008] One of the primary safety concerns regarding breast implants is implant rupture. A rupture is a tear or hole in the outer shell of the breast implant that is not necessarily noticeable. In the case of saline-filled breast implants, the breast contracts as the saline is absorbed by the body. In the case of silicone gel-filled breast implants, rupture means that the gel is "silent" and remains within the shell or within the scar tissue that forms around the implant, or the gel migrates outward through the scar tissue, and in some cases, outward near the augmented or reconstructed breast.

[0009] Clinical complications resulting from leaked silicone filler gel typically manifest as granulomas (small inflammatory nodules) and axillary lymphadenopathy (enlarged lymph nodes in the axillary area) (Holmich et al., (2004). Untreated Silicone Breast Implant Rupture Plastic and Reconstructive Surgery. 114 (1): 204-214; Katzin et al., (2005). Pathology of Lymph Nodes from Patients with Breast Implants: A Histologic and Spectroscopic Evaluation American Journal of Surgical Pathology. 29 (4): 506-11; FDA Breast Implant Consumer Handbook - Study of Rupture of Silicone Gel-filled Breast Implants (MRI Component) - 2004). Rupture rates increase with increasing implant duration. Overall, the rupture rate is generally less than 5% by the fourth year and increases approximately four to six years after implantation. After six years, the rupture rate continues to increase variably. Rupture resolves with explantation (surgical removal) of the implant.

[0010] In 2016, the World Health Organization (WHO) designated breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) as a T-cell lymphoma that can arise from breast implants (Swerdlow et al., (2016) The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood, 127(20), 2375-2390). Clinically, BIA-ALCL typically arises in the peri-breast implant capsule and presents as a fluid collection or tumor adjacent to the implant surface (FDA Executive Summary Breast Implant Special Agenda, prepared for the General and Plastic Surgery Devices Advisory Committee Meeting, March 25-26, 2019). As of July 6, 2019, the Food and Drug Administration (FDA) had received a total of 573 U.S. and global medical device reports (MDRs) for BIA-ALCL, including 33 deaths ("Medical Device Reports for Breast Implant-Associated Anaplastic Large Cell Lymphoma," www.fda.gov., as of July 24, 2019).

[0011] Collagen is the main component of connective tissue and the most abundant protein in mammals, accounting for approximately 30% of the total body protein. Collagen functions as the main component and major structural and mechanical determinant of the extracellular matrix (ECM) of most tissues (see, e.g., Kadler K. Birth Defects Res C Embryo Today. 2004; 72:1-11; Kadler KE, Baldock C, Bella J, Boot-Handford RP. J Cell Sci. 2007; 120:1955-1958; Kreger ST. Biopolymers. 2010 93(8): 690-707).

[0012] Due to its unique characteristics and diverse profile of functions in the human body, collagen is often chosen from a variety of biocompatible materials used to repair tissue, support structural integrity, guide cell infiltration, and promote tissue regeneration. Of the five major collagen types, type I collagen is the most abundant form in the human body. Collagen's unique properties make it a popular choice for regenerative medicine products.

[0013] Additive manufacturing (AM) is generally the process of producing three-dimensional (3D) objects using a computer model of the object. The basic operation of an AM system consists of slicing the 3D computer model into thin cross sections, converting the results into 2D positional data, and feeding the data to a controller that produces the 3D structure layer by layer.

[0014] Various AM techniques exist, including stereolithography, digital light processing (DLP), and three-dimensional (3D) printing (such as 3D inkjet printing). These techniques generally involve the layer-by-layer deposition and curing (e.g., solidification) of one or more build materials, which typically include photopolymerizable (light-curable) materials.

[0015] For example, stereolithography is an additive manufacturing process that uses a liquid ultraviolet (UV)-curable build material and a UV laser. In such a process, a laser beam traces the cross-section of the part pattern on the surface of the dispensed liquid build material at each dispensed layer of build material. Exposure to the UV laser light cures and hardens the traced pattern in the build material, bonding it to the layer below. After building, the formed part is immersed in a chemical bath to remove excess build material and then cured in a UV oven.

[0016] For example, in a three-dimensional printing process, a dispensing head with a set of nozzles dispenses a build material to deposit layers onto a support structure, and depending on the build material, the layers can then be cured or solidified using an appropriate device.

[0017] Build materials can include a modeling material formulation and a support material formulation, which upon solidification form the object and a temporary support structure that supports the object under construction, respectively.

[0018] A modeling material formulation is deposited to produce the desired object, and a support material formulation, with or without modeling material elements, is used to provide support structures in specific areas of the object being built to ensure proper vertical alignment of subsequent object layers, for example, when the object includes overhanging features or shapes (e.g., curved shapes, negative angles, gaps, etc.).

[0019] Both the modeling material and the support material are preferably liquid at the dispensing operating temperature and then solidify upon exposure to solidification or curing conditions, typically curing energy (e.g., UV curing), to form the required layer shape. After printing is complete, the support structures (if present) are removed to reveal the final shape of the fabricated 3D object. Solidification (hardening) of the dispensed materials typically involves polymerization (e.g., photopolymerization) and / or crosslinking (e.g., photocrosslinking).

[0020] Additive manufacturing was first used in biological applications to create three-dimensional sacrificial resin molds from which 3D scaffolds were created from biological materials.

[0021] 3D bioprinting is an additive manufacturing method that uses biological materials, optionally combined with chemicals and / or cells, to print 3D structures layer by layer, with precise positioning and tight control of the placement of functional components.

[0022] Three-dimensional (3D) bioprinting is gaining momentum in many medical applications, particularly in regenerative medicine, to address the need for complex scaffolds, tissues, and organs suitable for transplantation.

[0023] Unique to 3D printing in general is that the mechanical properties of the print medium (the dispensed build material) can differ significantly from the cured (solidified) material after printing.

[0024] To allow for precise control of post-printing curing (e.g., polymerization), build materials typically contain polymerizable (e.g., photopolymerizable) moieties or groups that polymerize (e.g., by chain extension and / or crosslinking) upon dispensing, thereby preserving the geometric shape and imparting the desired physical and / or mechanical properties to the final product.

[0025] Various techniques have been developed for 3D bioprinting, including 3D inkjet printing, extrusion printing, laser printing, and projection stereolithography (see, e.g., Murphy SV, Atala A, Nature Biotechnology. 2014 32(8). Miller JS, Burdick J. ACS Biomater. Sci. Eng. 2016, 2, 1658-1661). Each technique imposes different requirements on the dispensed build material (also referred to herein as the print medium), resulting from the specific application mechanism and the curing / gelation process required to maintain the 3D structure of the printed scaffold.

[0026] For all techniques, the most important parameters determining printing accuracy and efficiency are the static and dynamic physical properties of the dispensed build material, such as viscosity, shear thinning, and thixotropy. The static and dynamic properties of the build material are not only important for printing technology, but also when considering cell-containing printing, i.e., cells within the build material dispensed during printing. In this case, the shear forces applied to the build material during printing (dispensing) have a significant impact on cell survival. Therefore, it is desirable to have good control over the specific properties of the print medium over a wide range of conditions, i.e., concentration, temperature, ionic strength, and pH.

[0027] Type I collagen is considered a good candidate for use as the main component of the build material in 3D bioprinting.

[0028] Collagen methacrylate can be used as a rapidly self-assembling type I collagen to form crosslinked hydrogels for tissue engineering [see, e.g., Isaacson et al., Experimental Eye Research 173, 188-193 (2018)]. Collagen methacrylate has been used with mesenchymal stem cells [Kathryn E. Drzewiecki et al., A thermoreversible, photocrosslinkable collagen bio-ink for free-form fabrication of scaffolds for regenerative medicine, Technology (2017)], fibroblasts, adipose-derived stem cells, epithelial cells, and many other cells. Collagen methacrylate is useful for forming scaffolds of varying stiffness by varying the collagen concentration and curing conditions (e.g., irradiation intensity and duration).

[0029] Tissue-extracted collagen methacrylate has been characterized for its utility in 3D bioprinting (extrusion, inkjet, and photolithography) [Drzewiecki, KE et al. Langmuir 30, 11204-11211 (2014); Gaudet, ID & Shreiber, DI Biointerphases 7, 25 (2012)].

[0030] Despite the significant advantages of this natural polymer, many factors hinder its use in 3D bioprinting. The use of tissue-extracted collagen for this purpose is limited by its sensitivity to temperature and ionic strength, which spontaneously forms a gel above 20 °C under physiological conditions [see, for example, PureCol from Advanced BioMatrix, Inc.]. The typical temperature-dependent gel formation of tissue-extracted collagen significantly hinders accurate flow during printing. A possible solution to this phenomenon is to keep the print medium at a low temperature until application, but this represents a significant technical limitation. Another solution is the use of gelatin, a denatured form of collagen that does not gel under these conditions. However, gelatin lacks the true tissue and cellular interactions of natural collagen, thereby missing important biological functions.

[0031] The present assignee has developed a technology that allows for the purification of native human type I collagen (rhcollagen) by incorporating five human genes encoding heterotrimeric type I collagen into tobacco plants (see, e.g., Stein H. (2009) Biomacromolecules; 10:2640-5). This protein is purified to homogeneity in a cost-effective industrial process that takes advantage of collagen's unique properties. See also WO 2006 / 035442, WO 2009 / 053985, WO 2011 / 064773, WO 2013 / 093921, WO 2014 / 147622, ​​and patents and patent applications derived therefrom, the contents of all of which are incorporated herein by reference in their entirety.

[0032] The assignee's WO 2018 / 225076 describes curable recombinant human collagen and kits containing same that can be used to prepare modeling material formulations for additive manufacturing of 3D objects (e.g., 3D bioprinting), and methods of utilizing such modeling material formulations in the additive manufacturing of 3D objects having at least a portion thereof made of collagen-based materials. The formulations are characterized by a desirable viscosity at temperatures above 10°C (e.g., room temperature or 37°C), allowing additive manufacturing to occur without cooling the system or any portion thereof.

[0033] The assignee's WO 2019 / 211854 describes light-curable (photo-initiated) dermal fillers, hyaluronic acid-rh collagen dual cross-linked dermal fillers, and hyaluronic acid-rh collagen semi-interpenetrating networks, each containing plant-derived human collagen, and methods of using the same.

[0034] Further background art includes U.S. Patent Nos. 5,591,444, 7,723,108, 7,745,105, 7,919,112, 8,025,869, 8,038,665, 8,066,691, 8,124,120, 8,142,815, 8,192,487, 8,435,600, 8,546,142, 8,702,684, and 8,641,775. , No. 8,642,735, No. 8,697,059, No. 8,992,551, No. 8,758,781, No. 8,778,333, No. 8,778,909, No. 8,871,267, No. 9,101, No. 692, No. 9,074,190, No. 9,173,975, No. 9,150,668, No. 9,289,533, No. 9,956,317, No. 9,681,941, No. 9,752,138, No. 9, No. 744,260, No. 9,782,517, No. 9,801,976, No. 9,901,440, No. 9,913,705, No. 9,956,072, No. 10,011,820, No. 10,258,58 No. 8, No. 10,117,822, No. 10,039,633, No. 10,300,169, No. 10,335,190, No. 10,327,884, No. 10,449,034, No. 10,471,181 No.; German Patent No. 102011121982; Russian Patent No. 2675019; European Patent Application Publication No. 2995278; European Patent Nos. 3013379, 2231061, 1280562, 3247413, 1814606, 2550028, 2841115, 1734894, 3191020, 1546307, 3357519;and U.S. Patent Application Publication Nos. 2017 / 0274052, 2011 / 0274666, 2018 / 0064854, 2020 / 0078411, 2020 / 0030495, 2019 / 0321158, 2018 / 0177917, 2019 / 0184064, 2017 / 0071725, 2020 / 0030496, 2019 / 0060516, Examples include specifications Nos. 2020 / 0016191, 2019 / 0374457, 2018 / 0193522, 2018 / 0098836, 2017 / 0224896, 2019 / 0134265, 2017 / 0087273, 2018 / 0289860, 2018 / 0015204, 2020 / 0268503 and 2021 / 0030528. Summary of the Invention

[0035] The present inventors have devised rh-collagen-containing formulations that can be used to form degradable implants and injectable fillers suitable for use in soft tissue augmentation, reconstruction, and / or regeneration procedures. The disclosed implants and injectable fillers are designed with controlled mechanical, physical, and / or biological properties to suit the intended use and can be utilized in a variety of applications (e.g., reconstruction or augmentation of a subject's breast tissue), as further described below.

[0036] According to one aspect of some embodiments of the present invention, there is provided a three-dimensional (3D) biocompatible and degradable soft tissue implant comprising a 3D bioprinted composite scaffold, the composite scaffold comprising recombinant human collagen (rhcollagen) and a biocompatible synthetic polymer; and A porous wall; an interior cavity at least partially enclosed within a porous wall; at least one injection port connecting the interior cavity with an outermost surface of the scaffold, the injection port having an opening sized to allow insertion of an injection device through said injection port; A soft tissue implant is provided, comprising:

[0037] According to some of the embodiments described herein, the scaffold further comprises at least one printed vascular network path connecting the outermost surface of the scaffold with an interior cavity of the scaffold, the vascular network path being sized to allow vascular cells and tissue to penetrate.

[0038] According to some of the embodiments described herein, the scaffold comprises between about 1 and 1000 printed vascular network channels.

[0039] According to some of the embodiments described herein, the total volume of the interior cavity is between about 5 mL and about 300 mL, or between about 10 mL and about 300 mL, or between about 50 mL and about 300 mL.

[0040] According to some of the embodiments described herein, the internal cavity includes at least one chamber.

[0041] According to some of the embodiments described herein, the internal cavity includes between 2 and 30 chambers.

[0042] According to some of the embodiments described herein, at least two of the chambers are interconnected with each other.

[0043] According to some of the embodiments described herein, the scaffold comprises 1 to 15 injection ports.

[0044] According to some of the embodiments described herein, a bioprinted composite scaffold is formed by bioprinting a curable composition in a configuration pattern corresponding to the desired shape and dimensions of the soft tissue implant, the curable composition comprising recombinant human collagen (rh collagen) having curable moieties.

[0045] According to some of the embodiments described herein, the curable formulation further comprises a biocompatible synthetic polymer having a curable moiety.

[0046] According to some of the embodiments described herein, the rh collagen hardenable portion and the synthetic polymer hardenable portion are hardenable when subjected to the same hardening conditions.

[0047] According to some of the embodiments described herein, each of the curable moieties is a photocurable moiety.

[0048] According to some of the embodiments described herein, the biocompatible synthetic polymer is polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), poly(N-isopropylacrylamide) (PNIPAAm), poly-4-hydroxybutyrate (P4HB), or a copolymer of any of these.

[0049] According to some of the embodiments described herein, the rh collagen comprises plant-derived recombinant human collagen.

[0050] According to some of the embodiments described herein, the ratio of rh collagen to biocompatible synthetic polymer is about 1:1 to about 1:20, or about 1:1 to 1:10, or about 1:2 to 1:10.

[0051] According to some of the embodiments described herein, the scaffold further comprises at least one extracellular matrix (ECM) component.

[0052] According to some of the embodiments described herein, the formulation includes at least one ECM component having a curable moiety.

[0053] According to some of the embodiments described herein, the at least one ECM component comprises at least one of hyaluronic acid, fibronectin, heparin, elastin, laminin, and any combination thereof.

[0054] According to some of the embodiments described herein, the scaffold further comprises an integrin-binding material.

[0055] According to some of the embodiments described herein, the integrin-binding material is an RGD-containing material.

[0056] According to some of the embodiments described herein, the formulation comprises an integrin-binding moiety having a curable moiety.

[0057] According to some of the embodiments described herein, the implant further comprises a matrix within at least the interior cavity of the scaffold, the matrix comprising at least one extracellular matrix (ECM) component and at least one of cells or adipose tissue.

[0058] According to some of the embodiments described herein, the at least one ECM component comprises rh collagen, hyaluronic acid (HA), fibronectin, heparin, elastin, or laminin, or any combination thereof.

[0059] According to some of the embodiments described herein, the rh collagen comprises at least one of crosslinked fibrillar rh collagen and rh collagen-derived nanoparticles.

[0060] According to some of the embodiments described herein, the matrix further comprises an integrin-binding material.

[0061] According to some of the embodiments described herein, the weight ratio of ECM components to cells or adipose tissue within the matrix ranges from 1:1 to 1:5.

[0062] According to some of the embodiments described herein, the cells include pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells, adipocytes, or any combination thereof.

[0063] According to some of the embodiments described herein, the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue.

[0064] According to some of the embodiments described herein, the cells comprise a minimally processed extract derived from adipose tissue.

[0065] According to some of the embodiments described herein, the volume of the matrix is ​​from about 5 mL to about 300 mL.

[0066] According to some of the embodiments described herein, the soft tissue implant is a breast implant.

[0067] According to some of the embodiments described herein, the soft tissue is selected from breast tissue, facial tissue, neck tissue, muscle tissue, joint tissue, jaw tissue, buttock tissue, hand tissue, and chest tissue.

[0068] According to one aspect of some embodiments of the present invention, there is provided a method of preparing an implant as described herein in any of the embodiments and any combination thereof, comprising dispensing at least one formulation to sequentially form a plurality of layers in an architectural pattern of a scaffold, Methods are provided in which, for at least a portion of the layer, dispensing is a formulation comprising recombinant human collagen having at least one curable group and a synthetic polymer having at least one curable group, and optionally an ECM component having a curable group and / or an integrin-binding material having at least one curable material.

[0069] According to some of the embodiments described herein, the method further comprises injecting a matrix described herein in any of the embodiments and any combination thereof into at least the interior cavity of the scaffold via the injection port.

[0070] According to an aspect of some embodiments of the present invention there is provided a kit comprising at least one formulation for forming a scaffold as described herein in any of the embodiments and any combination thereof, and an injectable matrix formulation for forming a matrix as described herein in any of the embodiments and any combination thereof, wherein the kit is specified for the preparation of a soft tissue implant.

[0071] According to one aspect of some embodiments of the present invention, there is provided a soft tissue implant as described herein in any of the embodiments and any combination thereof, for implementation thereof in a subject in need of soft tissue augmentation and / or reconstruction and / or regeneration, comprising implanting a scaffold into a body organ or cavity in which soft tissue augmentation and / or reconstruction is desired, and optionally injecting a matrix as described herein into at least the interior cavity of the scaffold; A soft tissue implant is provided in which the matrix is ​​injected either before or after implantation.

[0072] According to some of the embodiments described herein, matrix injection is performed after implantation and repeated as needed.

[0073] According to some of the embodiments described herein, the volume of the matrix is ​​from about 5 mL to about 300 mL.

[0074] According to some of the embodiments described herein, after implantation, the printed vascular network pathway is anastomosed with at least one of the subject's blood vessels.

[0075] According to one aspect of some embodiments of the present invention, there is provided an injectable matrix formulation comprising at least one extracellular matrix (ECM) component and cells or adipose tissue or a combination thereof, wherein the at least one ECM component comprises rh collagen, hyaluronic acid, fibronectin, heparin, elastin, laminin or any combination thereof, and the cells comprise pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells or adipocytes, or any combination thereof, and / or a fat fraction isolated from adipose tissue.

[0076] According to some of the embodiments described herein, the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue.

[0077] According to some of the embodiments described herein, the cells comprise a minimally processed fraction isolated from adipose tissue.

[0078] According to some of the embodiments described herein, the rh collagen comprises plant-derived human collagen.

[0079] According to some of the embodiments described herein, the rh collagen comprises crosslinked fibrillar rh collagen and / or particles of rh collagen (particulate rh collagen).

[0080] According to some of the embodiments described herein, the weight ratio of ECM components to cells or adipose tissue is about 5:1 to about 1:5.

[0081] According to some of the embodiments described herein, the formulation further comprises an integrin-binding material.

[0082] According to some of the embodiments described herein, the integrin-binding material is an RGD-containing material.

[0083] According to an aspect of some embodiments of the present invention there is provided a matrix formulation as described herein for use in reconstructing and / or augmenting and / or regenerating soft tissue in a subject in need thereof.

[0084] According to some of the embodiments described herein, the soft tissue includes tissue of the face, nose, chin, breast, chin, buttocks, hand, muscle, joint, leg, foot, chest, lip or cheek, or any combination thereof.

[0085] According to some of the embodiments described herein, the formulation is injected into the soft tissue in a volume of about 5 to about 200 mL, or about 50 mL to about 150 mL.

[0086] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not necessarily intended to be limiting.

[0087] When implementing the method and / or system of the present invention, the selection tasks may be performed or completed manually, automatically, or a combination thereof. Furthermore, depending on the actual device or apparatus of the method and / or system of the present invention, some selection tasks may be performed by hardware, software, or firmware, or a combination thereof using an operating system.

[0088] For example, hardware for performing a selection task according to embodiments of the present invention may be implemented as a chip or circuit. In the case of software, a selection task according to embodiments of the present invention may be implemented as a plurality of software instructions executed by a computer using any suitable operating system. In exemplary embodiments of the present invention, one or more tasks according to exemplary embodiments of the methods and / or systems described herein are performed by a data processor, such as a computing platform, executing a plurality of instructions. Optionally, the data processor includes volatile memory for storing instructions and / or data, and / or non-volatile storage, e.g., a magnetic hard disk and / or removable media, for storing instructions and / or data. Optionally, a network connection may also be provided. Optionally, a display and / or user input device (e.g., a keyboard or mouse) may also be provided.

[0089] Several embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. It is emphasized that the details shown below, with particular reference to the drawings, are by way of example and for the purpose of providing a detailed description of embodiments of the invention. In this regard, it will become apparent to those skilled in the art, from a reading of the description together with the drawings, how embodiments of the present invention may be practiced. [Brief explanation of the drawings]

[0090] [Figure 1] Figures 1A-1B (Background Art) show an example of a compression test between two parallel plates used to characterize the mechanical properties of an implant (adapted from Brandon et al. (2019) Bioengineering (Basel). 6(2):43, Figures 2A and 2B). Figure 1A shows an image of the implant between the plates before the compression test. Figure 1B shows the same implant undergoing the compression test. (D0 = implant diameter (cm) before compression at time 0, D = implant diameter, d = contact diameter; H = implant projection between the plates (plate spacing), F = applied force). [Figure 2] Figure 2 (Background Art) shows an exemplary graph of analytical test load as a function of projective strain (adapted from Brandon et al. (2019) Bioengineering (Basel). 6(2):43, Figure 11). The change in implant projection, i.e., plate displacement, is measured in response to compressive load. [Figure 3] Figure 3 (Background Art) shows an exemplary graph of analytical test load as a function of diametral strain (adapted from Brandon et al. (2019) Bioengineering (Basel). 6(2):43, Figure 12). The change in implant diameter is measured in response to compressive load. [Figure 4] Figure 4 (Background Art) shows an exemplary graph of analytical test load as a function of areal strain (adapted from Brandon et al. (2019) Bioengineering (Basel). 6(2):43, Figure 13). The change in implant surface area is measured in response to compressive load. [Figure 5] Figure 5 (Background Art) shows an image of an exemplary setup for analyzing local strain at very low compressive loads ("pinch" testing) (adapted from Brandon et al. (2019) Bioengineering (Basel). 6(2):43, Figure 17). The implant deformation results from a low, localized uniaxial compressive force applied to the implant's periphery. The leftmost image shows the Xf setup. The middle image shows the placement of the implant within the setup, and the rightmost image shows the implant clamped for force measurement. (Stop = predetermined stop; fixed and gauge fingers are silicon probes simulating fingertips; D0 = initial implant diameter; Xf = final pinch distance; F = pinch force). [Figure 6]Figure 6 (Background Art) shows an exemplary graph depicting the results obtained in a "pinch" test (adapted from Brandon et al. (2019) Bioengineering (Basel). 6(2):43, Figure 18). The results show the deformation of the implant due to a low, localized uniaxial compressive force applied to the periphery of the implant by the "fingers." [Figure 7] FIG. 7 shows a schematic diagram of the fabrication and use of a 3D bioprinted breast implant containing a degradable scaffold, according to an exemplary embodiment of the present invention. The degradable breast implant scaffold is prepared by bioprinting (top left), and a matrix containing ECM components, such as rh-collagen, and optionally mixed with stromal vascular fraction (SVF), homogenized adipose extract, and / or cellular components, is infused into the degradable scaffold (top right). The ECM components, in some embodiments, can include hyaluronic acid (HA), fibronectin, heparin, or laminin, or any combination thereof. The matrix-filled scaffold is then implanted in the body and vascularized (bottom left). The implant scaffold then gradually degrades and is gradually replaced by new tissue, including regeneration and vascularization of the new tissue (bottom right). At this final stage, the scaffold disappears, leaving behind fully functional vascularized tissue. [Figure 8] Figures 8A-8D show images of a dome-shaped (10 mm x 10 mm x 6 mm) 3D-printed degradable scaffold (containing 0.5 mm x 0.5 mm pores) according to an exemplary embodiment of the present invention. Cross-sectional view (Figure 8A), top view (Figure 8B), and side view (Figure 8C) are shown. The two recesses visible in the top and side views are injection ports that can be filled with the matrices described herein before, after, or both before and after implantation. Figure 8D shows a 3D-bioprinted degradable scaffold containing methacrylated rh collagen (0.5%), PEG-DA (0.5-2%), SR9035 (1.0-2.5%), and HEAA (12.0-40.0%). [Figure 9]Figure 9 shows a schematic diagram of an exemplary method for isolating functional cells from the stromal vascular fraction (SVF), which can be mixed with an ECM matrix (e.g., a rh collagen-containing matrix) to load a degradable scaffold. The illustrated steps include isolating a fat pad from a subject (an adult rat), mechanically mincing the fat pad, enzymatically digesting the tissue to remove adipose components, and then recovering the SVF, which may contain pericytes, adipose-derived stem cells, preadipocytes, endothelial and progenitor cells, and / or hematopoietic cells, including monocytes and macrophages. In some embodiments, the isolation procedure is halted by mechanical mincing / homogenization of the fat pad without enzymatic digestion, resulting in a minimally processed homogenized fat extract. [Figure 10] 10 shows a schematic diagram of an exemplary 3D bioprinted scaffold filled with SVF and rh collagen-based matrix according to an exemplary embodiment of the present invention, prior to implantation into a subject requiring downstream regeneration of breast tissue. The scaffold is filled by injection through the injection ports as shown. The exemplary scaffold includes three injection ports in (e.g., fluid) communication with the internal cavity. The bioprinted scaffold further includes printed vascular network pathways distributed around its periphery, with openings at the edges of the scaffold. [Figure 11] Figure 11 shows a schematic diagram of an exemplary animal (rat / mouse) study to evaluate the safety and efficacy of implanted scaffolds loaded with a matrix comprising at least one ECM component, such as rh-collagen, and adipose tissue extract (VSF). The 3D-printed dome-shaped scaffolds are implanted into the animals, and the matrix is ​​loaded into a syringe and injected into the implanted scaffold. At predetermined times after implantation, the implants are retrieved and evaluated histologically for safety and efficacy. [Figure 12] 12A-12C show schematics and photographs outlining another exemplary animal (rat / mouse) study to evaluate the safety and efficacy of implanted scaffolds loaded with a matrix comprising at least one ECM component, such as rh-collagen, and adipose extract (minimally processed homogenized adipose extract). The scaffolds were loaded with matrix (FIG. 12A) and then implanted (FIG. 12B), as shown in a representative animal photograph after implantation (FIG. 12C). [Figure 13-1]FIG. 13A is a bar graph showing the histological scoring of the tested implants 4 weeks after implantation in rats. [Figure 13-2] Figure 13B shows representative histological images of both implant types with examples of tissue reactions: neovascularization is indicated by broad arrows, connective tissue proliferation around the printed strands is indicated by regular arrows, triangles indicate tissue ingrowth, and fatty infiltration is indicated by asterisks. [Figure 13-3] Figures 13C-13D show further representative histological images of both implant types, along with examples of tissue response. Figure 13C shows tissue ingrowth observed within the (previously empty) inner compartment of the scaffold and between the scaffold strands. Figure 13D further illustrates neovascularization with broad arrows. [Figure 13-4] Figure 13E shows another representative histological image of both implant types, along with examples of tissue reactions. Figure 13E shows a representative image showing neovascularized tissue ingrowth and fatty infiltration between the scaffold strands. [Figure 14] 14A-14D show images of an exemplary design of a scaffold according to some embodiments of the present invention, designed to enhance tissue ingrowth. The 3D scaffold features several interconnected internal compartments and thin channels connecting the internal space to the surface. [Figure 15] 15A-15C show images of an exemplary compact 3D scaffold with pleats and a central section to increase surface area and enhance tissue ingrowth: top view (FIG. 15A) and side view (FIGS. 15B-15C). [Figure 16] FIG. 16 is a bar graph showing the effect of rh collagen-based injectable matrices on cell proliferation. DETAILED DESCRIPTION OF THE INVENTION

[0091] The present invention, in some embodiments thereof, relates to the repair and / or augmentation of soft tissue, and more particularly, but not exclusively, to collagen-based formulations that can be used to form degradable scaffolds and soft tissue implants comprising the same, and collagen-based formulations that can be used as soft tissue fillers and / or in combination with degradable scaffolds.

[0092] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components and / or methods illustrated in the following description and / or drawings and / or examples. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0093] The present inventors have successfully designed and implemented a method for forming a degradable (e.g., biodegradable) composite scaffold by an additive manufacturing process (e.g., 3D bioprinting) utilizing curable rh collagen with a curable synthetic material that hardens into a synthetic polymer, preferably a biocompatible synthetic polymer. As described in further detail below, the scaffold is designed to have porous walls, an internal cavity, one or more injection ports, and, optionally, a vascular network pathway connecting the outermost surface of the scaffold with the internal cavity of the scaffold. The composite structure, which forms part of a soft tissue implant, is designed to have mechanical, physical, and / or biological properties that allow and / or promote soft tissue growth at the implantation site, such that newly formed soft tissue replaces the degradable implant.

[0094] The inventors have further succeeded in designing and using injectable matrices comprising one or more ECM components (e.g., rh collagen, optionally in combination with one or more additional ECM components) and optionally biological materials such as cells and / or adipose tissue, which can be used as soft tissue fillers or injected (loaded) into the composite scaffolds of the present embodiments as part of a degradable implant.

[0095] 1A-6 illustrate background art methods and desired parameters that can be used to control and determine the mechanical and / or physical properties of implants and / or matrices according to some embodiments of the present invention, which can be used to engineer implants and / or matrices with desired properties (e.g., by controlling the chemical nature and relative amounts of the components that make up the composite structures and / or matrices described in any of the embodiments herein).

[0096] FIG. 7 shows a schematic diagram of the fabrication and use of an exemplary 3D bioprinted breast implant including a degradable scaffold, according to an exemplary embodiment of the present invention. The degradable breast implant scaffold is prepared by bioprinting (top left), and a matrix containing ECM components, such as rh-collagen, and optionally mixed with stromal vascular fraction (SVF), homogenized adipose extract, and / or cellular components, is infused into the degradable scaffold (top right). The ECM components, in some embodiments, can include hyaluronic acid (HA), fibronectin, heparin, or laminin, or any combination thereof. The matrix-filled scaffold is then implanted in the body and vascularized (bottom left). The implant scaffold then gradually degrades and is gradually replaced by new tissue, including regeneration and vascularization of new tissue (bottom right). At this final step, the scaffold disappears, leaving behind fully functional vascularized tissue.

[0097] 8A-8D, 10, 14A-14B and 15 show images of exemplary designs of 3D bioprinted degradable scaffolds according to exemplary embodiments of the present invention.

[0098] FIG. 9 shows an example of the production of a biological material (fat extract) that can be incorporated into an injectable matrix according to some embodiments of the present invention.

[0099] As shown in Figures 11-13E, the inventors have demonstrated in vivo studies that upon implantation of the degradable implants described herein, neovascularization and tissue ingrowth occurs at the implantation site.

[0100] As shown in Figure 16, the inventors have further demonstrated that the injectable matrices described herein promote tissue growth.

[0101] Accordingly, embodiments of the present invention relate to 3D bioprinted degradable composite scaffolds, curable formulations and additive manufacturing processes for preparing the same, soft tissue implants comprising the formulations, and injectable matrices that can be used by themselves or in combination with the scaffolds to promote tissue growth. Embodiments of the present invention further relate to the use of the composite scaffolds and / or injectable matrices for the reconstruction, augmentation, replacement, and / or regeneration of soft tissue in subjects in need thereof.

[0102] According to some embodiments of the present invention, the implants, composite scaffolds and injectable matrices use recombinant human collagen (rh collagen).

[0103] Embodiments of the present invention further relate to three-dimensional (3D) biocompatible and degradable soft tissue (e.g., breast) implants, matrices comprising extracellular matrix (ECM) components and cells or adipose tissue, and methods and uses thereof.

[0104] Biocompatible, degradable soft tissue (e.g., breast) implants according to some of the present embodiments include a composite scaffold that can be bioprinted to incorporate components including recombinant human collagen (rhcollagen) and a biocompatible synthetic polymer. The composite scaffold components can further include at least one extracellular matrix component (ECM) component, such as, but not limited to, rhcollagen, hyaluronic acid (HA), fibronectin, heparin, elastin, or laminin, or any combination thereof. The implant scaffold includes a porous lattice, an internal cavity, and at least one injection port sized to allow insertion of a cannula for cell or tissue injection. The implant scaffold can further include at least one printed vascular network pathway to allow vascular cells and tissue to enter the implant.

[0105] The soft tissue (e.g., breast) implants disclosed herein can be used in methods for implantation into a subject in need thereof (including, but not limited to, for the purpose of soft tissue (e.g., breast) reconstruction or augmentation), where the implant degrades over time and is replaced by new tissue. Methods for preparing degradable soft tissue (e.g., breast) implants are also disclosed.

[0106] Disclosed herein are matrices comprising at least one ECM component and cells or adipose tissue, or both. In some embodiments, the matrix is ​​injected into a soft tissue (e.g., breast) implant prior to implantation, while in other embodiments, the matrix is ​​injected into the implant after implantation into a subject. In some other embodiments, the matrix is ​​used to reconstruct or augment soft tissue in a subject, independent of the use of a composite scaffold.

[0107] Recombinant human collagen (rh collagen): Collagen is an important structural protein of the extracellular matrix (ECM) of various connective tissues in the body. Collagen is the most abundant protein in mammals and consists of polyamino acid chains that bind together to form triple helices in elongated fibers. Bones, tendons, and cartilage contain collagen, as do different structures such as the cornea, blood vessels, intestines, and teeth. Type I collagen is the most abundant collagen in the human body.

[0108] Those skilled in the art will understand that the term "collagen chain" can encompass collagen subunits such as the α1 or α2 chains of a collagen fiber, which can be a type I fiber. Those skilled in the art will understand that the term "collagen" can encompass the assembled collagen trimer, which in the case of type I collagen, includes an α1 chain or two α1 chains and one α2 chain. A collagen fiber is collagen that lacks the terminal propeptides C and N.

[0109] One of the hallmarks of collagen is the regular arrangement of amino acids in each of the three chains of the triple helix. The amino acid sequence often contains repeats of Gly-Pro-X or Gly-X-hydroxyproline, where X can be any of a variety of other amino acid residues. Typically, proline or hydroxyproline residues account for approximately one-sixth of the total sequence, and glycine residues account for one-third of the total sequence. Hydroxylation of proline and lysine residues by prolyl-4-hydroxylase (P4H) occurs within the Gly-XY repeat region as the polypeptide chain translocates cotranslationally across the endoplasmic reticulum (ER) membrane. P4H is an enzyme composed of two subunits, α and β, both of which are required to form the active enzyme. The stability of the final triple helix structure of collagen is highly dependent on P4H-mediated hydroxylation of the collagen chains. Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50), and glucosyltransferase (EC 2.4.1.66) are enzymes involved in the post-translational modification of collagen. They sequentially modify lysyl residues at specific positions to hydroxylysyl, galactosylhydroxylysyl, and glucosylgalactosylhydroxylysyl residues. A single human enzyme, lysyl hydroxylase 3 (LH3), can catalyze all three consecutive steps in the formation of hydroxylysine-linked carbohydrates.

[0110] In some embodiments described herein, the collagen used in the present invention comprises collagen selected from the group consisting of animal-derived collagen, recombinant human collagen (rh collagen), or plant-derived recombinant human collagen. In some embodiments, the collagen comprises cross-linkable plant-derived human collagen. In some embodiments, the collagen comprises native collagen. In some embodiments, the collagen comprises modified collagen. In some embodiments, the collagen is modified by adding functional groups. In some embodiments, the collagen comprises linked collagen. In some embodiments, the collagen comprises cross-linked collagen.

[0111] In some embodiments, the collagen comprises isolated naturally occurring collagen, which may be obtained from a collagen-containing tissue selected from the group consisting of tendons, ligaments, skin, cornea, cartilage, blood vessels, intestines, intervertebral discs, muscles, bones, or teeth.

[0112] In some embodiments described herein, the rh collagen comprises plant-derived recombinant human collagen. In some embodiments described herein, the plant-derived human collagen is obtained from a transgenic plant. Those skilled in the art will understand that the term "transgenic plant" can include any lower plant (e.g., moss) or higher (vascular) plant or tissue or isolated cell thereof (e.g., in a cell suspension) that has been stably or transiently transformed with at least one exogenous polynucleotide sequence. In some embodiments, the transgenic plant is selected from the group consisting of tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. In some of the embodiments described herein, the transgenic plants include tobacco plants as described in WO 2006 / 035442, WO 2009 / 053985, WO 2011 / 064773, WO 2013 / 093921, WO 2014 / 147622, ​​and WO 2018 / 225076.

[0113] In some embodiments, any type of collagen chain can be expressed by the transgenic plant. In some embodiments, the collagen chains include fibril-forming collagens (types I, II, III, V, and XI), network-forming collagens (types IV, VIII, and X), collagens associated with the fibril surface (types IX, XII, and XIV), collagens occurring as transmembrane proteins (types XIII and XVII), or collagens that form 11 nm periodic beaded filaments (type VI). In some embodiments, the expressed collagen chains are the α1 and / or α2 chains of type I collagen.

[0114] In some embodiments, the expressed collagen alpha 1 chain can be encoded by any polynucleotide sequence derived from any mammal. In some embodiments, the nucleic acid sequences encoding collagen alpha chains 1 and 2 are of human origin and are SEQ ID NOs: 1 and 2, respectively. In some embodiments, the transgenic plant comprises a sequence encoding the human collagen alpha-1 chain of SEQ ID NO: 1. In some embodiments, the transgenic plant comprises a sequence encoding the human collagen alpha-2 chain of SEQ ID NO: 2.

[0115] In some embodiments, the transgenic plant produces an amino acid sequence comprising the modified human collagen alpha-1 chain of SEQ ID NO: 3. In some embodiments, the transgenic plant produces an amino acid sequence encoding the modified human collagen alpha-2 chain of SEQ ID NO: 4.

[0116] In some embodiments, the transgenic plant comprises one or more sequences encoding P4H-α and P4H-β. In some embodiments, the exogenous P4H expressed by the transgenic plant comprises a mammalian P4H. In some embodiments, the exogenous P4H comprises a human P4H. In some embodiments, the transgenic plant comprises one or more sequences encoding the human P4H of SEQ ID NOs: 5 and 6.

[0117] In some embodiments, the transgenic plant comprises a sequence encoding a mammalian LH3. In some embodiments, the transgenic plant comprises a sequence encoding the LH3 of SEQ ID NO:7.

[0118] In some embodiments, the alpha collagen chains expressed in the plant may or may not include their terminal propeptides (i.e., propeptide C and propeptide N). In some embodiments, the transgenic plant includes sequences encoding protease N, protease C, or both.

[0119] In some other embodiments, the plant-derived human collagen can be a procollagen molecule containing partially digested propeptides. Furthermore, in some embodiments, the plant-derived human collagen comprises atelocollagen. Those skilled in the art will appreciate that the term "atelocollagen" can encompass collagen molecules lacking both the N-terminal and C-terminal propeptides. In some embodiments, the plant-derived human collagen is atelocollagen having an amino acid (AA) sequence derived from the collagen α-chain-encoding sequences of SEQ ID NO: 1 and SEQ ID NO: 2. Atelocollagen can be obtained by enzymatic digestion (e.g., digestion with ficin) of the procollagen product of SEQ ID NO: 1 and SEQ ID NO: 2.

[0120] In some embodiments, polynucleotide sequences encoding the alpha chain and / or modifying enzymes (e.g., P4H and LH3) can be modified in a way that alters their cellular localization when expressed in plants.

[0121] Those skilled in the art will be familiar with various methods for introducing nucleic acid constructs into transgenic plants, including, for example, direct DNA transfer, such as Agrobacterium-mediated gene transfer or electroporation. Those skilled in the art will also be familiar with various plant breeding techniques, and therefore, further description of such techniques will not be provided herein. In some embodiments, plant tissues / cells are harvested at maturity and collagen fibers are isolated using well-known extraction methods.

[0122] In some of the embodiments described herein, the recombinant human collagen is recombinant human type I collagen.

[0123] In some embodiments, type I collagen is considered the primary component of the build material in 3D bioprinting. In some embodiments, the soft tissue (e.g., breast) implant composite scaffold comprises type I collagen. In some embodiments, the soft tissue (e.g., breast) implant composite scaffold comprises recombinant type I collagen. In some embodiments, the soft tissue (e.g., breast) implant composite scaffold comprises recombinant human type I collagen. In some embodiments, the soft tissue (e.g., breast) implant composite scaffold comprises plant-produced recombinant human type I collagen.

[0124] In some embodiments, the matrices described herein comprise type I collagen. In some embodiments, the matrices described herein comprise recombinant type I collagen. In some embodiments, the matrices described herein comprise recombinant human type I collagen. In some embodiments, the matrices described herein comprise recombinant human type I collagen produced in a plant.

[0125] In some of the embodiments described herein, the recombinant human collagen is plant-derived recombinant human collagen, and in some embodiments, the plant is tobacco.

[0126] In some embodiments described herein, plant-derived human collagen is prepared using a system for purifying type I recombinant human collagen (rhcollagen), which involves introducing five human genes encoding heterotrimeric type I collagen into tobacco plants (COLLPLANT™, Israel). The unique properties of collagen are exploited to purify the protein to homogeneity through a cost-effective industrial process. See also WO 2006 / 035442, WO 2009 / 053985, and patents and patent applications derived therefrom, the entire contents of all of which are incorporated herein by reference.

[0127] In some of the embodiments described herein, the human recombinant collagen (rh collagen) described in any of the embodiments herein is monomeric rh collagen.

[0128] By "monomeric" it is meant that the rh collagen described herein is soluble in aqueous solutions and does not form fibrillar aggregates.

[0129] In some of the embodiments described herein, the human recombinant collagen (rh collagen) described in any of the embodiments herein is fibrillar rh collagen, which is also referred to herein as "crosslinked rh collagen" or "crosslinked fibrillar rh collagen."

[0130] By "fibrillar," it is meant that the rh collagen described herein takes the form of fibrillar aggregates in an aqueous solution containing fibrillar aggregates. Typically, but not necessarily, fibrillar rh collagen is formed by exposing monomeric rh collagen to a fibrillogenesis buffer (usually characterized by a basic pH). An exemplary procedure for forming fibrillar rh collagen is described in the Examples section below.

[0131] In some of the embodiments described herein, the rh collagen is particulate rh collagen, e.g., in the form of rh collagen-derived nanoparticles, which may include, e.g., rh collagen nanoparticles and / or rh gelatin nanoparticles formed as degradation products during preparation of the nanoparticles, as described in the Examples section below.

[0132] In some embodiments described herein, the rh collagen is a curable rh collagen as described herein. In some embodiments described herein, the rh collagen is formed from curable rh collagen, which is formed by subjecting the curable rh collagen, alone or in combination with other curable materials, to curing conditions as described in any of the embodiments herein.

[0133] Typically, but not necessarily, the curable rh collagen described herein is used to form the composite scaffolds of the present invention, and fibrous and / or particulate rh collagen is used to form the soft tissue fillers of the present invention.

[0134] In some embodiments, type I collagen is used to prepare 3D degradable soft tissue (e.g., breast) implants comprising a composite scaffold. In some embodiments, recombinant type I collagen is used to prepare 3D degradable soft tissue (e.g., breast) implants comprising a scaffold. In some embodiments, recombinant human type I collagen is used to prepare 3D degradable soft tissue (e.g., breast) implants comprising a scaffold. In some embodiments, plant-produced recombinant human type I collagen is used to prepare 3D degradable soft tissue (e.g., breast) implants comprising a scaffold. In some embodiments, plant-produced recombinant human cross-linked type I collagen is used to prepare 3D degradable soft tissue (e.g., breast) implants comprising a scaffold. In some embodiments, plant-produced recombinant human modified type I collagen (e.g., the hardenable plant-produced recombinant human type I collagen described herein) is used to prepare 3D degradable soft tissue (e.g., breast) implants comprising a scaffold.

[0135] Bioprinted composite scaffolds: According to an aspect of some embodiments of the present invention there is provided a composite scaffold comprising recombinant human collagen (rhcollagen) and a biocompatible synthetic polymer, as described in any of the embodiments herein.

[0136] According to some of the embodiments described herein, the composite scaffold is a bioprinted composite scaffold formed by additive manufacturing (e.g., 3D bioprinting) in a configuration pattern that essentially corresponds to the shape of the implant that contains it, as described herein.

[0137] According to some of the embodiments described herein, the composite scaffold is a 3D bioprinted composite scaffold.

[0138] Those skilled in the art will appreciate that the term "scaffold" as used herein can encompass three-dimensional (3D) structures used to enhance or promote cell growth and tissue formation.

[0139] According to some embodiments described herein, a composite scaffold (e.g., a 3D bioprinted composite scaffold) is characterized by a porous lattice, an internal cavity within the scaffold, and at least one injection port connecting the internal cavity to the outermost surface of the scaffold. According to some embodiments described herein, a scaffold includes a porous lattice forming a porous wall, an internal cavity at least partially enclosed within the porous wall, and at least one injection port connecting the internal cavity to the outermost surface of the scaffold.

[0140] According to some of the embodiments described herein, at least one or each of the injection ports has an opening sized to allow an injection device to be inserted therethrough.

[0141] According to some embodiments described herein, the scaffolds disclosed herein are comprised of a highly porous, artificial, three-dimensional network of interconnected pores that are used in vivo as a framework to which cells can attach. In instances where the scaffold comprises a matrix (as described in further detail below), both cells contained in the matrix and additional cells can grow on and within the scaffold to regenerate tissue as needed. In some embodiments, the scaffolds described herein can be considered living scaffolds that can be formed to contain living cells. One or more living cells can be attached to the scaffold. The living cells can be grown for a period of time during which the cells grow and form colonies, which then fuse to form a cellular network and subsequently form living tissue within the scaffold. In some embodiments, the scaffold promotes cell growth in three dimensions.

[0142] In some embodiments, the composite scaffold provides a suitable surface for cell adhesion and proliferation. The composite scaffold can further provide mechanical stability and support. The composite scaffold can have a particular shape or form that influences or defines the three-dimensional shape or form assumed by the growing cell population. Such shapes or forms include, but are not limited to, domes, cubes, cones, spheres, rolls, rectangles, three-dimensional amorphous shapes, etc.

[0143] Those skilled in the art will understand that the term "pore" can encompass voids or spaces in the composite scaffold. In some embodiments, the pores comprise a uniform, interconnected porous network. In some embodiments, the pores comprise a non-uniform, interconnected porous network. In some embodiments, the interconnected porous network provides space for cells to grow and proliferate, nutrients to reach them, and waste products to be removed by diffusion. In some embodiments, at least one printed vascular network pathway provides space for vascular tissue and cells, which can provide nutrients to the cells within the porous network. In some embodiments, the pore openings are sized to allow cell entry into the porous network of the scaffold.

[0144] According to some of the embodiments described herein, the composite scaffold comprises a porous lattice in which solid spaces form and surround a plurality of pores, hi some embodiments, the solid spaces are designed to provide support, shape, and structure to the scaffold or an implant comprising it.

[0145] In some embodiments, the solid space can comprise a porous network, such as when the scaffold comprises a porous network. In some embodiments, the solid space can comprise a lattice, such as when the scaffold comprises a porous lattice. In some embodiments, the solid space can comprise a braided lattice, such as when the scaffold comprises a braided lattice. In some embodiments, the solid space can comprise a woven lattice, such as when the scaffold comprises a woven lattice. In some embodiments, the solid space can comprise a series of struts. In some embodiments, the solid space can comprise a series of filaments. In some embodiments, the solid space can comprise a series of walls. In some embodiments, the solid space can comprise a combination of filaments, struts, walls, woven lattice, or braided lattice. In some embodiments, the filaments, struts, walls, woven lattice, or braided lattice can comprise tubular, square, rectangular, triangular, or freeform shapes. In some embodiments, such filaments, struts, walls, woven lattice, or braided lattice can be curved. In some embodiments, such filaments, struts, walls, woven lattice, or braided lattice can be straight. In some embodiments, such filaments, struts, walls, woven lattice, or braided lattice can be regularly distributed within the scaffold. In some embodiments, such filaments, struts, walls, woven or braided lattices can be randomly distributed within the scaffold. In some embodiments, such filaments, struts, walls, woven or braided lattices can be regularly distributed in some areas and randomly distributed in other areas within the scaffold. In some embodiments, the filaments, struts, walls, woven or braided lattices of the composite scaffold form a porous wall or walls and are not present in an internal cavity.

[0146] In some embodiments, the thickness of the filaments, struts, lattices, or walls of the composite scaffold is about 1 μm to about 5 μm, about 1 μm to about 10 μm, about 5 μm to about 10 μm, about 5 μm to about 25 μm, about 5 μm to about 50 μm, about 10 μm to about 50 μm, about 10 μm to about 75 μm, about 10 μm to about 100 μm, about 25 μm to about 100 μm, or about 50 μm to about 100 μm. In some embodiments, the thickness of the filaments, struts, lattices or walls of the composite scaffold is from about 1 μm to about 1000 μm, from about 1 μm to about 500 μm, from about 100 μm to about 1000 μm, from about 50 μm to about 500 μm, from about 500 μm to about 1000 μm, from about 50 μm to about 250 μm, from about 100 μm to about 500 μm, from about 100 μm to about 750 μm, from about 250 μm to about 750 μm, from about 250 μm to about 1000 μm, or from about 50 μm to about 750 μm (including intermediate values ​​and subranges therebetween).

[0147] In some embodiments, the thickness of the filaments, struts, lattices or walls of the composite scaffold is at least 1 μm, at least 2 μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 6 μm, at least 7 μm, at least 8 μm, at least 9 μm, at least 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, or at least 100 μm. In some embodiments, the thickness of the filaments, struts, lattices or walls of the composite scaffold is at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, at least 1000 μm, at least 250 μm, at least 350 μm, at least 450 μm, at least 550 μm, at least 650 μm, at least 750 μm, at least 850 μm, at least 950 μm, or at least 150 μm.

[0148] In some embodiments, the pores in the porous network, lattice, or wall of the scaffold are uniformly spaced. In some embodiments, the pores in the porous network, lattice, or wall of the scaffold have a repeating pattern. In some embodiments, the pores in the porous network, lattice, or wall of the scaffold vary in shape and size. In some embodiments, the pores in the porous network, lattice, or wall are uniform in size.

[0149] In some embodiments, the pores in the porous network, lattice, or walls of the scaffold have the same shape. In some embodiments, the pores in the porous network, lattice, or walls of the scaffold are not the same shape. In some embodiments, the shape of the pores is not limited and can be any number of 3D shapes. In some embodiments, the pore shape includes a polyhedron. In some embodiments, the pore shape includes a cube, a prism, or a pyramid. In some embodiments, the pore shapes include, but are not limited to, a circle, a square, a rectangle, an ellipse, a parallelogram, a triangle, a dodecahedron (such as a pentagonal dodecahedron), a 3D kagome, a diamond, and an octahedron. In some embodiments, the pore shape promotes cell migration, cell proliferation, cell differentiation, and tissue growth.

[0150] In some embodiments, when the pore shape is square or rectangular, the pore dimensions are about 50 μm to 200 μm by about 50 μm to 1000 μm, or 50 μm by 50 μm by 100 μm, or 50 μm by 150 μm, or 50 μm by 200 μm, or 50 μm by 250 μm, or 50 μm by 300 μm, or 50 μm by 350 μm, or 50 μm by 400 μm, or 50 μm by 450 μm, or 50 μm by 500 μm, or 50 μm by 550 μm, or 50 μm by 600 μm, or 50 μm by 650 μm, or 50 μm by 700 μm, or 50 μm by 750 μm, or 50 μm by 600 μm. In some embodiments, the pore size is about 100 μm×100 μm, or 100 μm×150 μm, or 100 μm×200 μm, or 100 μm×250 μm, or 100 μm×300 μm, or 100 μm×350 μm, or 100 μm×400 μm, or 100 μm×450 μm, or 100 μm×1000 μm, or is 100 μm×550 μm, or 100 μm×600 μm, or 100 μm×650 μm, or 100 μm×700 μm, or 100 μm×750 μm, or 100 μm×800 μm, or 100 μm×850 μm, or 100 μm×900 μm, or 100 μm×950 μm, or 100 μm×1000 μm. In some embodiments, the pore size is about 150 μm×150 μm, or 150 μm×200 μm, or 150 μm×250 μm, or 150 μm×300 μm, or 150 μm×350 μm, or 150 μm×400 μm, or 150 μm×450 μm, or 150 μm×500 μm, or 150 μm×550 μm, or 150 μm×600 μm, or 150 μm×650 μm, or 150 μm×700 μm, or 150 μm×750 μm, or 150 μm×800 μm, or 150 μm×850 μm, or 150 μm×900 μm, or 150 μm×950 μm, or 150 μm×1000 μm.In some embodiments, the pore size is about 200 μm×200 μm, or 200 μm×250 μm, or 200 μm×300 μm, or 200 μm×350 μm, or 200 μm×400 μm, or 200 μm×450 μm, or 200 μm×1000 μm.

[0151] In some embodiments, the pore openings are sized to allow cellular penetration into the porous network of the scaffold. In some embodiments, the pore openings or pore diameters are between about 50 μm and 800 μm. In some embodiments, the pore diameters are between about 10 μm and 100 μm. In some embodiments, the pore diameters are between about 100 μm and 700 μm. In some embodiments, the pore diameters are between about 150 μm and 600 μm. In some embodiments, the pore diameters are between about 200 μm and 550 μm. In some embodiments, the pore diameters are between about 250 μm and 500 μm. In some embodiments, the pore diameters are between about 300 μm and 450 μm. In some embodiments, the pore diameters are between about 350 μm and 400 μm.

[0152] In some embodiments, the average diameter of the pores is about 10 μm, or 20 μm, or 30 μm, or 40 μm, or 50 μm, or 60 μm, or 70 μm, or 80 μm, or 90 μm, or 100 μm, or 120 μm, or 140 μm, or 160 μm, or 180 μm, or 200 μm, or 220 μm, or 240 μm, or 260 μm, or 280 μm, or 300 μm, or 320 μm, or 340 μm. μm, or 360 μm, or 380 μm, or 400 μm, or 420 μm, or 440 μm, or 460 μm, or 480 μm, or 500 μm, or 520 μm, or 540 μm, or 560 μm, or 580 μm, or 600 μm, or 620 μm, or 660 μm, or 680 μm, or 700 μm, or 720 μm, or 740 μm, or 760 μm, or 780 μm, or 800 μm.

[0153] Throughout this specification, "μm" means micrometer, also referred to interchangeably herein as "micron."

[0154] According to some of the embodiments described herein, the composite scaffold comprises a porous lattice forming a porous wall, or a plurality of walls, e.g., interconnected walls forming a porous network, each wall being made up of a plurality of pores, as described herein in any of the embodiments and any combination thereof.

[0155] As used herein, the terms "porous lattice," "porous wall," and "porous network" are used interchangeably to describe the portions of a scaffold that at least partially surround an internal cavity as described herein.

[0156] According to some embodiments described herein, the scaffold has a rough surface. In some embodiments, the average surface roughness (Ra) is about 0.025 micrometers or greater, e.g., about 0.025 to about 50 micrometers, or to about 20 micrometers. Surface roughness can be engineered, for example, by folded porous walls, as described in more detail below (see Figures 15A-15C).

[0157] In some other embodiments, the scaffold has a smooth surface.

[0158] In some embodiments, the scaffold has a dome shape. In some embodiments, the scaffold has a freeform shape. In some embodiments, the size and shape of the scaffold corresponds to its intended use, for example, the size and shape of the scaffold is determined by the desired size and shape of an implant containing the scaffold, which is determined, inter alia, by the anatomy of the subject.

[0159] In some embodiments, the volume of the scaffold is about 1 to about 5 mL, or about 5 mL to about 20 mL, or about 20 to about 100 mL, or about 100 to about 500 mL, or about 50 to about 300 mL (including intermediate values ​​and subranges therebetween).

[0160] In some embodiments, the open space within the scaffold includes at least one cavity (e.g., an internal cavity described herein) and pores. In some embodiments, the total open space within the scaffold has a volume of about 1 to about 5 mL, or about 5 mL to about 20 mL, or about 20 to about 100 mL, or about 100 to about 500 mL, including intermediate values ​​and subranges therebetween. In some embodiments, the total volume of the open space within the scaffold is greater than 500 mL.

[0161] In some embodiments, the volume of total open space within the scaffold network (e.g., the total volume of pores and internal cavities) is about 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 50 mL, 60 mL, 70mL, 80mL, 90mL, 100mL, 110mL, 120mL, 130mL, 140mL, 150mL, 160mL, 170mL, 180mL, 190mL, 200mL, 210mL, 220mL, 230mL, 240mL, 250mL, 260mL, 270mL, 280mL, 290mL, 300mL, 350mL, 400mL, 450mL, or 500mL.

[0162] In some embodiments, the volume of the scaffold is at least 1 mL, at least 2 mL, at least 3 mL, at least 4 mL, at least 5 mL, at least 6 mL, at least 7 mL, at least 8 mL, at least 9 mL, at least 10 mL, at least 11 mL, at least 12 mL, at least 13 mL, at least 14 mL, at least 15 mL, at least 16 mL, at least 17 mL, at least 18 mL, at least 19 mL, at least 20 mL, at least 25 mL, at least 30 mL, at least 35 mL, at least 40 mL, at least 50 mL, at least 60 mL, at least 70 mL, at least 80 mL, at least 90 mL, at least 100 mL, at least 110 mL, at least 120 mL, at least 130 mL, at least 140 mL, at least 150 mL, at least 160 mL, at least 170 mL, at least 180 mL, at least 190 mL, at least 200 mL, at least 210 mL, at least 220 mL, at least 230 mL, at least 240 mL, at least 250 mL, at least 260 mL, at least 270 mL, at least 280 mL, at least 290 mL, at least 300 mL, at least 350 mL, at least 400 mL, at least 450 mL, or at least 500 mL.

[0163] The scaffolds described herein include an internal cavity therein, eg, an internal cavity at least partially surrounded by a porous wall as described herein.

[0164] In some embodiments, the volume of the internal cavity is about 5 mL to about 300 mL, or about 50 mL to about 250 mL, or about 100 mL to about 200 mL (including intermediate values ​​and subranges therebetween). In some embodiments, the volume of the internal cavity is about 20 mL to about 50 mL. In some embodiments, the volume of the internal cavity is about 50 mL to 150 mL. In some embodiments, the volume of the internal cavity is about 150 mL to 300 mL.

[0165] In some embodiments, the volume of the interior cavity is about 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 210 mL, 220 mL, 230 mL, 240 mL, 250 mL, 260 mL, 270 mL, 280 mL, 290 mL, 300 mL, or 350 mL. In some embodiments, the volume of the interior cavity is at least 50 mL, at least 60 mL, at least 70 mL, at least 80 mL, at least 90 mL, at least 100 mL, at least 110 mL, at least 120 mL, at least 130 mL, at least 140 mL, at least 150 mL, at least 160 mL, at least 170 mL, at least 180 mL, at least 190 mL, at least 200 mL, at least 210 mL, at least 220 mL, at least 230 mL, at least 240 mL, at least 250 mL, at least 260 mL, at least 270 mL, at least 280 mL, at least 290 mL, at least 300 mL, or at least 350 mL.

[0166] According to some embodiments described herein, the internal cavity comprises at least one chamber. As used herein, the term "chamber" encompasses a fully or partially enclosed space or compartment within a scaffold (e.g., within a porous lattice, network, or wall), which may, for example, comprise a matrix described herein. In some embodiments, the internal cavity comprises up to 20 chambers. In some embodiments, the internal cavity comprises up to 30 chambers. In some embodiments, the internal cavity comprises 1-30 chambers, or 2-30 chambers. In some embodiments, the internal cavity comprises 2-25 chambers. In some embodiments, the internal cavity comprises 2-15 chambers. In some embodiments, the internal cavity comprises 10-30 chambers. In some embodiments, the internal cavity comprises 10-20 chambers. In some embodiments, the internal cavity comprises 15-30 chambers.

[0167] In some embodiments, the internal cavity comprises 1 chamber, or 2 chambers, or 3 chambers, or 4 chambers, or 5 chambers, or 6 chambers, or 7 chambers, or 8 chambers, or 9 chambers, or 10 chambers, or 11 chambers, or 12 chambers, or 13 chambers, or 14 chambers, or 15 chambers, or 16 chambers, or 17 chambers, or 18 chambers, or 19 chambers, or 20 chambers, or 21 chambers, or 22 chambers, or 23 chambers, or 24 chambers, or 25 chambers, or 26 chambers, or 27 chambers, or 28 chambers, or 29 chambers, or 30 chambers. In some embodiments, the interior cavity comprises at least 1 chamber, at least 2 chambers, at least 3 chambers, at least 4 chambers, at least 5 chambers, at least 6 chambers, at least 7 chambers, at least 8 chambers, at least 9 chambers, at least 10 chambers, at least 11 chambers, at least 12 chambers, at least 13 chambers, at least 14 chambers, at least 15 chambers, at least 16 chambers, at least 17 chambers, at least 18 chambers, at least 19 chambers, at least 20 chambers, at least 21 chambers, at least 22 chambers, at least 23 chambers, at least 24 chambers, at least 25 chambers, at least 26 chambers, at least 27 chambers, at least 28 chambers, at least 29 chambers, or at least 30 chambers.

[0168] In some embodiments, when there is more than one chamber in the internal cavity, the chambers may always be different in size and / or shape from one another, or all the chambers may have the same size and / or shape.

[0169] In some embodiments, whenever there are two or more chambers in the internal cavity, at least two, and optionally preferably all, of the chambers are interconnected, e.g., by one or more channels or tunnels or other hollow structures that interconnect two (e.g., adjacent) chambers. In some other embodiments, the chambers are not directly connected, but are connected (e.g., allowing fluid communication between them) by a porous network around their periphery. In some embodiments, several chambers in the internal cavity of the scaffold are interconnected. In some of these embodiments, at least two, at least some, or all of the chambers are at least in fluid communication with each other, e.g., by one or more channels or tunnels or other hollow structures that interconnect two adjacent chambers and allow the flow of fluid (e.g., blood and / or its components) therebetween. In some embodiments, two or more chambers are interconnected, allowing the flow of fluid (e.g., liquid) containing cells and / or other biological components therebetween.

[0170] In some of the embodiments described herein, the total volume of the internal cavities is from about 10 to about 90% of the total volume of the scaffold, including any intermediate values ​​and subranges therebetween. In some embodiments, the total volume of the internal cavities is about 10 to about 80%, or about 10 to about 70%, or about 10 to about 60%, or about 10 to about 50%, or about 10 to about 40%, or about 10 to about 30%, or about 10 to about 20%, or about 20 to about 90%, or about 20 to about 80%, or about 20 to about 70%, or about 20 to about 60%, or about 20 to about 50%, or about 20 to about 40%, or about 20 to about 30%, or about 30 to about 90%, or about 30 to about 90%, or about 30 to about 80%, or about 30 to about 70%, or about 30 to about 60%, or about 30 to about 50% of the total volume of the scaffold. or about 30 to about 40%, or about 20 to about 90%, or about 20 to about 80%, or about 30 to about 70%, or about 30 to about 60%, or about 30 to about 50%, or about 30 to about 40%, or about 40 to about 90%, or about 40 to about 80%, or about 40 to about 70%, or about 40 to about 60%, or about 40 to about 50%, or about 50 to about 90%, or about 50 to about 80%, or about 50 to about 70%, or about 50 to about 60%, or about 60 to about 90%, or about 60 to about 80%, or about 60 to about 70%, or about 70 to about 90%, or about 70 to about 80%, or about 80 to about 90%.

[0171] In some of the embodiments described herein, the scaffolds described herein comprise at least one injection port connecting an internal cavity with the outermost surface of the scaffold (e.g., the outermost surface of the porous lattice, network, or wall). Those skilled in the art will understand that the term "injection port" refers to a hole, opening, or orifice that allows for the injection of a substance, such as a cell, tissue, or matrix, as detailed herein.

[0172] In some embodiments, the injection port opening is sized to allow insertion of a needle or cannula for cell or tissue injection and / or matrix injection, as described in any of the embodiments herein.

[0173] In some embodiments, the injection port has a diameter that allows injection using an appropriate injection device. In some embodiments, such devices include, but are not limited to, needles, cannulas, sharpened plastic tip applicators, reservoirs, stents, plungers, delivery systems, and syringes. In exemplary embodiments, the device has a maximum 20-gauge or maximum 18-gauge needle, and the diameter of the injection port is designed accordingly.

[0174] In some embodiments, the injection port has a diameter that allows for injection of a matrix, as described in detail herein, hi some embodiments, the injection port has a diameter that allows for injection of ECM components, cells and / or tissue, as described herein.

[0175] In some embodiments, the injection port has a diameter of about 0.3 mm to about 3 mm (including intermediate values ​​and subranges therebetween). In some embodiments, the injection port has a diameter of about 0.2 mm to about 2.5 mm. In some embodiments, the injection port has a diameter of about 0.5 mm to about 1 mm. In some embodiments, the injection port has a diameter of about 0.5 mm to about 2 mm.

[0176] In some embodiments, the injection port has a diameter of 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, or 3 mm. In some embodiments, the injection port has a diameter of at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm, at least 1.0 mm, at least 1.2 mm, at least 1.3 mm, at least 1.4 mm, at least 1.5 mm, at least 1.6 mm, at least 1.7 mm, at least 1.8 mm, at least 1.9 mm, at least 2.0 mm, at least 2.1 mm, at least 2.2 mm, at least 2.3 mm, at least 2.4 mm, at least 2.5 mm, at least 2.6 mm, at least 2.7 mm, at least 2.8 mm, at least 2.9 mm, or at least 3 mm.

[0177] In some embodiments, the matrix is ​​loaded into the scaffold prior to implantation into the subject, where the injection is performed through at least one injection port, and in some embodiments, the matrix is ​​injected into the scaffold using at least one injection port in the scaffold after implantation of the scaffold into the subject.

[0178] In some embodiments, the scaffold comprises up to 20 injection ports. In some embodiments, the scaffold comprises up to 30 injection ports. In some embodiments, the scaffold comprises 1-20 injection ports (including intermediate values ​​and subranges therebetween). In some embodiments, the scaffold comprises 10-30 injection ports. In some embodiments, the scaffold comprises 1-30 injection ports (including intermediate values ​​and subranges therebetween). In some embodiments, the scaffold comprises 5-25 injection ports. In some embodiments, the scaffold comprises 1-15 injection ports. In some embodiments, the scaffold comprises 20-30 injection ports. In some embodiments, the scaffold comprises 10-20 injection ports. In some embodiments, the scaffold comprises 15-30 injection ports.

[0179] In some embodiments, the scaffold comprises 1 injection port, 2 injection ports, 3 injection ports, 4 injection ports, 5 injection ports, 6 injection ports, 7 injection ports, 8 injection ports, 9 injection ports, 10 injection ports, 11 injection ports, 12 injection ports, 13 injection ports, 14 injection ports, 15 injection ports, 16 injection ports, 17 injection ports, 18 injection ports, 19 injection ports, or 20 injection ports. In some embodiments, the scaffold comprises at least 1 injection port, at least 2 injection ports, at least 3 injection ports, at least 4 injection ports, at least 5 injection ports, at least 6 injection ports, at least 7 injection ports, at least 8 injection ports, at least 9 injection ports, at least 10 injection ports, at least 11 injection ports, at least 12 injection ports, at least 13 injection ports, at least 14 injection ports, at least 15 injection ports, at least 16 injection ports, at least 17 injection ports, at least 18 injection ports, at least 19 injection ports, or at least 20 injection ports.

[0180] In some of the embodiments described herein, the scaffold includes at least one vascular network pathway connecting at least a portion of the outermost surface of the scaffold with the lumen of the scaffold. According to some of these embodiments, the vascular network pathway is a printed vascular network pathway, i.e., bioprinted or otherwise fabricated by additive manufacturing as described herein in conjunction with a composite scaffold as described herein. According to some of these embodiments, the vascular network pathway is sized to allow infiltration of vascular cells and tissue. In some embodiments, the vascular network pathway is configured to promote vascularization of the scaffold when an implant containing the vascular network is implanted in a subject. Specifically, the vascular network pathway is configured to promote growth of a target vascular network into the scaffold. Vascularization of the scaffold promotes cell and tissue growth within the implant. The target vascular network efficiently supplies oxygen and other nutrients to cells and tissue within the scaffold, such as those detailed herein. Furthermore, the target vascular network provides an efficient point of release for cellular waste and debris, allowing for efficient removal of cellular waste and / or debris from the area of ​​the implant. In some embodiments, the vascular network pathway is designed and configured to allow anastomosis to the subject's blood vessels via a surgical procedure.

[0181] In some embodiments, when a 3D bioprinted degradable implant described herein is implanted in a subject, the printed vascular network pathways can be surgically connected to the subject's blood vessels by anastomosis. In some embodiments, the subject's blood vessels connect through at least one of the printed vascular network pathways so that cells and tissues within the interior cavity of the scaffold receive a constant supply of oxygen and nutrients.

[0182] In some embodiments, the scaffold comprises up to 1,000 vascular network pathways. In some embodiments, the scaffold comprises 1 to about 1,000 printed vascular network pathways (including intermediate values ​​and subranges therebetween). In some embodiments, the scaffold comprises about 10 to about 1,000 vascular network pathways. In some embodiments, the scaffold comprises 1 to about 200 vascular network pathways. In some embodiments, the scaffold comprises 200 to 500 vascular network pathways. In some embodiments, the scaffold comprises 800 to 1,000 vascular network pathways. In some embodiments, the scaffold comprises 500 to 800 vascular network pathways. In some embodiments, the number of vascular network pathways is 1 to about 100. In some embodiments, the number of vascular network pathways is about 100 to about 200. In some embodiments, the number of vascular network pathways is about 200 to about 300. In some embodiments, the number of vascular network pathways is about 300 to about 400. In some embodiments, the number of vascular network pathways is about 400 to about 500. In some embodiments, the number of vascular network pathways is about 500 to about 600. In some embodiments, the number of vascular network pathways is about 600 to about 700. In some embodiments, the number of vascular network pathways is about 700 to about 800. In some embodiments, the number of vascular network pathways is about 800 to about 900. In some embodiments, the number of vascular network pathways is about 900 to about 1000.

[0183] Referring now to FIG. 8B, an exemplary 3D scaffold 10 according to some embodiments of the present disclosure is shown. In the illustrated embodiment, which should not be considered limiting, the scaffold 10 is dome-shaped, although other shapes are contemplated. The illustrated dome-shaped 3D scaffold 10 has lateral dimensions of approximately 10 mm x 10 mm and a height of approximately 6.0 mm, although other dimensions are contemplated. The scaffold 10 is preferably porous. In the illustrated embodiment, which should not be considered limiting, the pores 12 of the scaffold 10 are square pores, although other shapes are contemplated. In this exemplary scaffold, the dimensions of the pores 12 are approximately 0.5 mm x 0.5 mm, although other dimensions are contemplated.

[0184] In some embodiments, the scaffold 10 comprises a porous network (e.g., including but not limited to, a lattice, an internal cavity at least partially surrounded by the lattice, and at least one injection port connecting the internal cavity to the outermost surface of the scaffold), where the injection port opening is sized to allow insertion of an injection device (e.g., including but not limited to, a cannula for cell or tissue injection, etc.). In some embodiments, the scaffold comprises an internal cavity within the scaffold, as described in any of the embodiments herein.

[0185] A representative example of a scaffold 10 in an embodiment employing an internal cavity is shown schematically in FIG. 10 . Walls 14 of the scaffold 10 are shown, which are preferably porous, and the internal cavity 16 is at least partially enclosed within the walls 14. While the internal cavity 16 shown in FIG. 10 is in the form of multiple connecting lobes, other shapes for the internal cavity 16 are contemplated according to some embodiments of the present invention. Several injection ports 18 are also shown. While three injection ports are shown in FIG. 10 , the scaffold 10 can include any number of injection ports as described herein, including configurations lacking any ports. At least one of the injection ports 18 is sized to receive an injection device 20 for injecting material into the cavity 16. While FIG. 10 shows the injection device 20 as a syringe, any suitable device for injecting material can be used, including, but not limited to, the cannulas and pipettes described herein. Typically, the injection device 20 injects the matrix described herein into the cavity 16. As a non-limiting example shown in FIG. 10, device 20 injects SVF in a rh collagen-based matrix.

[0186] In some embodiments of the present invention, as described herein in any of the embodiments and any combination thereof, the wall 14 of the scaffold 10 comprises a vascular network 22. The vascular network 22 locally connects the outermost surface 24 of the wall 14 with the interior cavity 16. Preferably, the vascular network 22 is sized to allow vascular components, cells, and tissue to enter the cavity 16.

[0187] Another configuration suitable for this embodiment is shown in Figures 14A-D, which show a side view (Figure 14A), a top view (Figure 14B), a perspective view (Figure 14C), and a representative image (Figure 14D) of scaffold 10. In this configuration, scaffold 10 includes multiple internal cavities 16a, 16b, and 16c, which are preferably interconnected. The total volume of the scaffold shown in Figures 14A-D is typically about 160 mL to about 180 mL, e.g., about 168 mL. The total volume of internal cavities 16a, 16b, and 16c is typically about 30% to about 50%, e.g., about 40%, of the total volume of the scaffold. For example, if the total volume of the scaffold is about 168 mL, the total volume of the internal cavities can be about 49 mL.

[0188] A further exemplary embodiment is shown in Figures 15A-15C, which are schematic diagrams of a top view (Figure 15A) and a side view (Figures 15B-15C) of a scaffold 10. In this embodiment, the scaffold 10 includes folds (shown in dashed lines) that increase the surface area of ​​the scaffold, e.g., to enhance tissue ingrowth. In Figure 15A, the cavity 16 is shown in the center, although embodiments in which the cavity 16 is not central are also contemplated. The scaffold shown in Figures 15A-15C has lateral dimensions of approximately 10 mm x 10 mm and a height of approximately 5 mm.

[0189] Preparation of bioprinted composite scaffolds: According to some of the embodiments described herein, the 3D composite scaffolds described herein are formed by additive manufacturing, e.g., 3D bioprinting. The 3D composite scaffolds (also referred to throughout this specification as simply "scaffolds" or "composite scaffolds", or bioprinted scaffolds, or bioprinted composite scaffolds, or 3D bioprinted composite scaffolds, or 3D bioprinted scaffolds) include composite scaffolds formed by additive manufacturing processes, such as bioprinting, as described herein.

[0190] According to some of the embodiments described herein, a bioprinted composite scaffold is formed by bioprinting a curable formulation as described herein in a configuration pattern that generally corresponds to the desired shape and dimensions of a soft tissue implant comprising the composite scaffold, the composite scaffold comprising a porous lattice or wall, an internal cavity, one or more injection ports, and optionally a vascular network pathway, as described herein in any one or any combination of embodiments.

[0191] In some embodiments, bioprinting of 3D scaffolds involves a manufacturing process using a bioink formulation containing biological components with a manufacturing system such as digital light processing (DLP), stereolithography (SLA), inkjet printer, laser printer or extrusion printer, bioprinter or bioprinting system. In some embodiments, the 3D scaffold design is converted into a printable digital format such as stereolithography (STL) that is used for printing. One of skill in the art would be familiar with available technologies and commercially available bioprinters that support bioprinting of the 3D scaffolds and implants disclosed herein.

[0192] Those skilled in the art will appreciate that a bioprinted 3D composite scaffold is a form comprising a porous lattice, an internal cavity within the scaffold, and at least one injection port connecting the internal cavity to the outermost surface of the scaffold, the injection port being sized to allow insertion of a cannula for cell or tissue injection. The characteristics of the porous lattice, the internal cavity, the vascular network pathways, and the at least one injection port have been described in detail above and are fully incorporated herein (e.g., including but not limited to, shape, size, biological properties, number of internal cavity chambers, cavity volume, number of ports, and pore size).

[0193] According to one aspect of some embodiments of the present invention, there is provided a process (method) for additive manufacturing (AM) of three-dimensional composite scaffolds as described herein in any of the embodiments and any combination thereof. According to embodiments of this aspect, the method is carried out by sequentially forming multiple layers to form a composite scaffold as described herein in any of the embodiments and any combination thereof. According to embodiments of this aspect, each layer is formed by dispensing at least one uncured build material and exposing the dispensed build material to curing conditions to form a solidified (cured) material that constitutes at least a portion of the composite scaffold.

[0194] In some embodiments, the method of preparing a 3D bioprinted composite scaffold further comprises the step of subjecting the dispensed layer to curing conditions, e.g., by irradiating the composite scaffold with a light source, as described in further detail below.

[0195] Throughout this specification, the phrase "build material" includes the phrase "uncured build material" or "uncured build material formulation," which collectively describes materials dispensed in successive layers as described herein. This phrase includes the uncured materials that form the final object (composite scaffold), i.e., one or more uncured modeling material formulations, and in some cases also includes the uncured materials used to form the support, i.e., uncured support material formulations. Build materials also include non-curing materials that preferably do not undergo (or will not undergo) any change during the process, such as biological materials or components (other than the curable collagen described herein) and / or other agents or additives described herein.

[0196] The build material dispensed to form successive layers as described herein is also referred to interchangeably herein as a "print medium" or "bioprinting medium" or "bioink."

[0197] The uncured build material can include one or more modeling material formulations, and the uncured build material can be dispensed such that different portions of the object are formed upon solidification (e.g., curing) of different modeling formulations, and thus different portions are formed of different solidified (e.g., hardened) modeling materials or different mixtures of solidified (e.g., hardened) modeling materials.

[0198] In the method of this embodiment, a three-dimensional composite scaffold is fabricated layer by layer by forming multiple layers in a configuration pattern that corresponds to the desired shape, size, and all other characteristics of the scaffold, as described herein.

[0199] Each layer is formed by an additive manufacturing device that scans a two-dimensional surface to pattern it. During the scan, the device directs the device to multiple target locations on the two-dimensional layer or surface and, according to a preset algorithm, determines for each target location or group of target locations whether that target location or group of target locations is occupied with build material and what type of build material to deliver there. This determination is made according to a computer image of the surface.

[0200] When AM is performed by three-dimensional inkjet printing, uncured build material, as defined herein, is dispensed from a dispensing head having a set of nozzles, depositing the build material in layers onto a support structure. Thus, the AM device dispenses build material at target locations to be occupied, leaving other target locations free. The device typically has multiple dispensing heads, each configured to dispense a different build material (e.g., a different modeling material formulation (each comprising a different biological component, or a different curable material, or a different concentration of curable material) and / or a different support material formulation). Thus, different target locations can be occupied with different build materials (e.g., a modeling formulation and / or a support formulation, as defined herein).

[0201] The final three-dimensional object (composite scaffold) is formed from the solidified modeling material, or a combination of solidified modeling materials, or a combination of solidified modeling materials and support materials, or variations thereof (e.g., after curing). All such operations are well known to those skilled in the art of additive manufacturing (also known as solid freeform molding).

[0202] In some exemplary embodiments of the invention, a composite scaffold is fabricated by dispensing a build material comprising two or more different modeling material formulations, each dispensed from a different dispensing head of an AM device. The modeling material formulations are preferably deposited in layers as they pass through the dispensing heads as needed. The modeling material formulations and / or combinations of formulations within a layer are selected according to the desired properties of the object (composite scaffold).

[0203] An exemplary process according to some embodiments of the present invention begins by receiving 3D printing data corresponding to the shape, size, and all other characteristics of the composite scaffold, as described herein. The data can be received, for example, from a host computer transmitting digital data relating to manufacturing instructions based on computer object data, which can be in the form of, for example, Standard Tessellation Language (STL) or Stereolithography Contour (SLC) format, Virtual Reality Modeling Language (VRML), Additive Manufacturing File (AMF) format, Drawing Interchange Format (DXF), Polygon File Format (PLY), Digital Imaging and Communications in Medicine (DICOM), or any other format suitable for computer-aided design (CAD).

[0204] The process continues by dispensing build material as described herein in layers onto a receiving medium using one or more dispensing (eg, print) heads according to print data.

[0205] Depending on the additive manufacturing method used and the configuration selected, dispensing can occur in the form of droplets or a continuous stream.

[0206] The receiving medium can be a tray of a printing system, or a support or medium formed of or coated with a biocompatible material, such as a support medium or article commonly used in bioprinting, or a pre-deposited layer.

[0207] In some embodiments, the receiving medium includes a sacrificial hydrogel or other biocompatible material as a mold for embedding the print object, which is then removed by chemical, mechanical, or physical (e.g., heating or cooling) means. Such sacrificial hydrogels can be formed, for example, from pluronic materials or gelatin.

[0208] Once the uncured build material has been dispensed onto the receiving medium according to the 3D data, the method optionally and preferably continues with solidification of the dispensed formulation. In some embodiments, the process continues by exposing the deposited layers to curing conditions. Preferably, the application of curing conditions to each layer occurs after the deposition of that layer and before the deposition of the previous layer.

[0209] As used herein, the term "curing" refers to the process by which a formulation solidifies. Solidification of a formulation is typically accompanied by an increase in the viscosity of the formulation and / or an increase in the storage modulus (G') of the formulation. In some embodiments, a formulation dispensed as a liquid solidifies to a solid or semi-solid (e.g., a gel). A formulation dispensed as a semi-solid (e.g., a soft gel) solidifies to a solid or to a harder or stronger semi-solid (e.g., a strong gel).

[0210] As used herein, the term "cure" includes, for example, polymerization of monomeric and / or oligomeric materials and / or crosslinking of polymer chains (crosslinking of polymers present prior to curing or crosslinking of polymeric materials formed upon polymerization of monomers or oligomers). Thus, the product of a curing reaction is typically a polymeric and / or crosslinked material. As used herein, the term also includes partial curing, e.g., at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70% curing of the formulation, as well as 100% curing of the formulation.

[0211] The phrases "conditions affecting cure" or "conditions for inducing cure," herein interchangeably referred to as "cure conditions" or "cure-inducing conditions," refer to conditions that, when applied to a formulation containing a curable material, induce cure as defined herein. Such conditions can include, for example, the application of cure energy to the curable material, as described below, and / or contact of the curable material with chemically reactive components (e.g., catalysts, co-catalysts, and activators).

[0212] When the conditions that induce curing include the application of curing energy, the phrase "expose to curing conditions" and its grammatical variations means exposing the dispensed layer to curing energy, and the exposure is typically achieved by applying curing energy to the dispensed layer.

[0213] "Curing energy" typically includes the application of radiation and / or the application of heat.

[0214] The radiation can be electromagnetic radiation (e.g., ultraviolet or visible light), or electron beam radiation, or ultrasonic radiation, or microwave radiation, depending on the material to be cured. The radiation is applied (or irradiated) by a suitable radiation source. For example, as described herein, an ultraviolet light source, a visible light source, an infrared light source, a xenon light source, a mercury light source, a lamp light source, or an LED light source can be used.

[0215] Curable materials or systems that harden upon exposure to radiation are referred to interchangeably herein as "photopolymerizable" or "photoactivatable" or "photohardenable."

[0216] When the curing energy includes heat, curing is also referred to herein and in the art as "thermal curing" and involves the application of thermal energy. As described herein, the application of thermal energy can be accomplished, for example, by heating the receiving medium into which the layer is dispensed or a chamber containing the receiving medium. In some embodiments, heating is accomplished using a resistive heater.

[0217] In some embodiments, heating is achieved by irradiating the dispensed layer with thermally inducing radiation, which can be achieved, for example, by an IR lamp or a xenon lamp that is activated to emit radiation onto the deposited layer.

[0218] In some embodiments, heating is achieved by infrared radiation from a ceramic lamp, for example, a ceramic lamp that provides infrared radiation between about 3 μm and about 4 μm (eg, about 3.5 μm).

[0219] Curable materials or systems that harden upon exposure to heat are referred to herein as "thermosetting" or "thermoactivatable" or "thermopolymerizable."

[0220] In some of the embodiments described herein, solidifying the dispensed formulation comprises exposing the dispensed formulation to curing conditions, e.g., irradiation (illumination), as described in any of the embodiments herein.

[0221] In some embodiments, exposure to curing conditions occurs for a short period of time, e.g., less than 3 minutes, less than 300 seconds, e.g., from 10 seconds to 240 seconds, or from 10 seconds to 120 seconds, or from 10 seconds to 60 seconds, including any intermediate values ​​and subranges therebetween.

[0222] In some of the embodiments described herein, if a support material formulation is included in the build material, the method further includes exposing the hardened modeling material formulation to post-treatment conditions before or after removing the support material formulation. The post-treatment conditions are typically intended to further solidify the hardened modeling material. In some embodiments, the post-treatment solidifies a partially cured formulation to obtain a fully cured formulation.

[0223] In some embodiments, the post-treatment is by exposure to heat or radiation, as described in any of the embodiments herein.

[0224] In some embodiments, it is contemplated that different formulations may be dispensed from different dispensing heads to produce a composite scaffold, and such embodiments provide, among other things, the ability to select from a predetermined number of formulations and define the desired combination of selected formulations and their properties.

[0225] According to this embodiment, the spatial location of deposition of each formulation in the layer is determined such that different formulations occupy different three-dimensional spatial locations, or such that two or more different formulations occupy substantially the same three-dimensional location or adjacent three-dimensional locations, allowing for spatial combination of the formulations within the layer after deposition.

[0226] Thus, in this embodiment, a wide range of material combinations can be deposited, and objects can be produced in which different portions of the object are made from different combinations of multiple modeling material formulations according to the properties desired to characterize each portion of the object. Systems utilized in additive manufacturing may include a receiving medium and one or more dispensing heads. The receiving medium may be, for example, a manufacturing tray that may have a horizontal surface that carries material dispensed from a print head. In some embodiments, the receiving medium is formed from or coated with a biocompatible material, as described herein.

[0227] The dispensing head can be, for example, a print head having a plurality of dispensing nozzles arranged in one or more arrays along a longitudinal axis of the dispensing head, and the dispensing head can be oriented such that its longitudinal axis is substantially parallel to the indexing direction.

[0228] The additive manufacturing system can further include a controller, such as a microprocessor, that controls the AM process, for example, the operation of the dispensing head according to a predetermined scan plan (e.g., a CAD configuration converted into Standard Tessellation Language (STL) format and programmed into the controller). The dispensing head can include multiple jetting nozzles. The jetting nozzles dispense material onto a receiving medium to form layers representing a cross-section of the 3D object.

[0229] In addition to the dispensing head, a source of curing energy can be provided to cure the dispensed build material. The curing energy is typically radiation, such as ultraviolet or thermal radiation. Alternatively, means for providing curing conditions other than electromagnetic or thermal radiation can be provided, such as cooling the dispensed build material or contacting the build material with a reagent that promotes curing.

[0230] Additionally, the AM system may include a leveling device to level and / or define the height of each layer after deposition and at least partial solidification and before depositing the next layer.

[0231] According to this embodiment, the additive manufacturing methods described herein are for bioprinting biological objects.

[0232] As used herein, "bioprinting" means performing an additive manufacturing process using one or more curable (e.g., modeling) formulations (bio-ink formulations) that include biological components as described herein in a manner compatible with an automated or semi-automated computer-aided additive manufacturing system (e.g., a bioprinter or bioprinting system) as described herein.

[0233] Throughout this specification, the phrase "modeling material formulation" (also referred to interchangeably herein as "modeling formulation" or "modeling material composition" or "modeling composition," or simply "formulation" or "composition") refers to some or all of the uncured build material (print medium) that is dispensed to form the final object (composite scaffold), as described herein. A modeling formulation is an uncured, curable modeling formulation that forms the object (composite scaffold) or a portion thereof upon exposure to curing conditions.

[0234] In the context of bioprinting, the uncured build material comprises at least one modeling formulation that includes one or more biological components or materials (e.g., the curable rh collagen described herein), also referred to herein and in the art as a "bioink" or "bioink formulation."

[0235] In some embodiments, bioprinting involves the sequential formation of multiple layers of uncured build material in a configuration pattern, preferably according to three-dimensional printing data as described herein. At least one, and preferably most or all, of the formed layers comprises (before solidifying or curing) one or more biological components as described herein (e.g., curable rh collagen as described herein). Optionally, at least one of the formed layers comprises (before solidifying or curing) one or more non-biologic, curable materials and / or non-curable biological or non-biological components, preferably biocompatible materials that do not interfere with (e.g., do not adversely affect) the biological and / or structural characteristics of the biological components (e.g., collagen) in the print medium and / or bioink.

[0236] In some embodiments, the components in the bio-ink or print medium, e.g., the non-curable and curable materials, and / or the curing conditions applied to effect curing, are selected so as not to significantly affect the structural and / or functional properties of the biological components in the bio-ink or print medium.

[0237] In some of the embodiments described herein, the build material (e.g., print medium) comprises a modeling material formulation (bioink) and optionally a support material formulation, all of which are selected to comprise a material or combination of materials that do not interfere with the biological and / or structural characteristics of the biological component.

[0238] In some of the embodiments described herein, the bioprinting methods are configured to form layers under conditions that do not significantly affect the structural and / or functional properties of the biological components in the bio-ink.

[0239] In some embodiments, bioprinting systems for carrying out the bioprinting processes / methods described herein are configured to allow layers to be formed under conditions that do not significantly affect the structural and / or functional properties of the biological components in the bio-ink.

[0240] In some of the embodiments described herein, additive manufacturing (e.g., bioprinting) processes and systems are configured such that process parameters (e.g., temperature, shear force, shear strain rate) do not interfere with (substantially affect) the functional and / or structural characteristics of the biological components.

[0241] In some of the embodiments described herein, the additive manufacturing process (bioprinting) is carried out at a temperature of at least 10°C or at least 20°C, for example, at a temperature in the range of about 10 to about 40°C, preferably about 10°C to 37°C, or about 20°C to 37°C, or about 20°C to about 30°C, or about 20°C to about 28°C, or about 20°C to about 25°C (including intermediate values ​​and subranges therebetween), or room temperature, or 37°C.

[0242] In some of the embodiments described herein, the temperatures / temperature ranges mentioned above are the temperatures at which the build material (e.g., the modeling material formulation including at least the biological components described herein) is dispensed, i.e., the temperature of the dispensing head in the AM system and / or the temperature at which the modeling material formulation is held before passing through the dispensing head.

[0243] In some of the embodiments described herein, the AM process is performed without cooling the AM system (e.g., the dispensing head and / or the modeling material formulation) to a temperature below room temperature, e.g., below 20°C or below 10°C, or below 5°C (e.g., 4°C).

[0244] In some of the embodiments described herein, the AM system does not have a means to cool the system or portions thereof (e.g., the dispensing head and / or the modeling material formulation) to a temperature below room temperature, e.g., below 20°C or below 10°C, or below 5°C (e.g., 4°C).

[0245] In some of the embodiments described herein, additive manufacturing processes (bioprinting) are performed while applying shear forces that do not adversely affect the structural and / or functional properties of biological components (e.g., cells). The application of shear forces can be performed by passing a build material (e.g., a modeling material formulation including at least a biological component described herein) through a dispensing head, which should also be considered to subject the build material to shear forces.

[0246] According to some embodiments, the AM bioprinting process is carried out under conditions (e.g., low shear and room or physiological temperature) that do not affect the functional and / or structural characteristics of the biological components contained in the bioink, while maintaining the necessary fluidity (viscosity that confers fluidity, e.g., less than 10,000 centipoise, or less than 5,000 centipoise, or less than 2,000 centipoise) and further maintaining the hardenability of the dispensed build material.

[0247] The bioprinting method and corresponding system can be any of the methods and systems known in the art for additive manufacturing, examples of which are described above. Suitable methods and systems can be selected taking into account printing capabilities such as resolution, deposition rate, scalability, bioink compatibility, and ease of use.

[0248] For example, a suitable bioprinting system typically includes a dispensing system (either with a temperature control module or at room temperature), a stage (receiving medium), and movement along the x-, y-, and z-axes as directed by CAD-CAM software. The system may also include a curing source (e.g., a light source or heat source) and / or a humidifier that applies curing energy (e.g., applied light or thermal radiation) or conditions to the deposition area (receiving medium) to promote hardening of the formed layer. Printers exist that use multiple dispensing heads to facilitate sequential dispensing of several materials.

[0249] Generally, bioprinting can be performed using known techniques for additive manufacturing. Some exemplary additive manufacturing techniques are listed below, but any other techniques are not contemplated. do.

[0250] 3D Inkjet Printing: 3D inkjet printing is a common 3D printer used for both non-biological and biological (bioprinting) applications. Inkjet printers use thermal or acoustic forces to eject droplets onto a substrate that can support or form part of the final structure. This technique delivers a controlled amount of liquid to a predetermined location, resulting in high-resolution printing with precise control over (1) the ink droplet position and (2) the ink volume (which is beneficial for printing microstructures or when adding small amounts of bioreactive agents or drugs). Inkjet printers can be used with several inks, including, for example, multiple biological components and / or bioactive agents. Furthermore, printing is fast and applicable to culture plates.

[0251] Bioprinting methods utilizing 3D inkjet printing systems can be performed using one or more bio-ink modeling material formulations described herein, dispensing droplets of the formulation in layers onto a receiving medium using one or more inkjet printheads according to 3D printing data.

[0252] Extrusion printing: This technique uses continuous beads of material rather than droplets. These beads of material are deposited in 2D, with the stage (receiving medium) or extrusion head moving along the z-axis, with each deposited layer serving as the foundation for the next layer. The most common methods of extruding biological materials for 3D bioprinting applications are pneumatic or mechanical dispensing systems.

[0253] Stereolithography (SLA) and Digital Light Processing (DLP): SLA and DLP are additive manufacturing techniques that convert uncured build material in a bath into solidified material layer by layer by selectively curing it using a light source, which is then later separated / washed away from the solidified material. SLA is widely used in various industries, including bioprinting, to create models, prototypes, patterns, and production parts.

[0254] Laser printing: The type of laser-based printing technology employed in 3D bioprinting is based on the principle of laser-induced forward transfer (LIFT), which was developed to transfer metals and has now been successfully applied to biological materials. The device consists of a laser beam, a focusing system, an energy absorption / transduction layer, a biological material layer (e.g., cells and / or hydrogel), and a receiving substrate. Laser-based printers work by irradiating the absorption layer with a laser beam, which converts the energy into a mechanical force that extrudes small droplets from the biological layer onto the substrate. A light source is then used to harden the material on the substrate.

[0255] Laser printing is compatible with a range of viscosities, allowing mammalian cells to be printed without affecting cell viability or function. Cell deposition can be achieved at up to 10 8 This can be done at a density of 1 cell / mL and with microscale resolution of one cell per drop.

[0256] Electrospinning: Electrospinning is a fiber manufacturing technique that uses electrical forces to draw electrically charged threads of a polymer solution or melt.

[0257] Curable formulation: According to some of the embodiments described herein, the composite scaffold is formed by bioprinting one or more curable modeling material formulations and, optionally, support material formulations according to the embodiments described herein above with respect to additive manufacturing processes.

[0258] For convenience, one or more components of a curable modeling formulation are described herein as components of a curable formulation, but it should be understood that the components can be divided into two or more curable modeling material formulations according to a selected construction pattern described herein.

[0259] According to some of the embodiments described herein, the hardenable composition comprises a hardenable collagen, the collagen as described herein in any of the embodiments and any combination thereof.

[0260] According to some of the embodiments described herein, the hardenable formulation comprises recombinant human collagen (rh collagen) (also referred to herein as hardenable rh collagen) characterized by one or more hardenable moieties or groups.

[0261] "Curable," as used herein, means a material that is capable of hardening or solidifying, as defined herein, upon exposure to appropriate curing conditions.

[0262] Curable materials typically harden or harden through polymerization and / or crosslinking.

[0263] Curable materials are typically polymerizable materials that polymerize and / or crosslink upon exposure to appropriate curing conditions or appropriate curing energy (a suitable energy source). Alternatively, curable materials are thermoresponsive materials that harden or solidify upon exposure to a temperature change (e.g., heating or cooling). In some cases, curable materials are made of small particles (e.g., nanoparticles or nanoclays) that can harden to form a solidified material. In yet other cases, curable materials are biological materials that undergo a biological reaction (e.g., an enzyme-catalyzed reaction) to form a hardened or solid material.

[0264] In some of the embodiments described herein, the curable material is a photopolymerizable material that polymerizes and / or crosslinks upon exposure to radiation as described herein, and in some embodiments, the curable material is a UV-curable material that polymerizes or crosslinks upon exposure to UV-visible light as described herein.

[0265] In some of the embodiments described herein, when the curable material is exposed to curing conditions (e.g., radiation, reagents), it polymerizes by chain extension, entanglement, and / or crosslinking. Crosslinking can be chemical and / or physical.

[0266] In some of the embodiments described herein, the curable material can be a monofunctional curable material or a multifunctional curable material.

[0267] As used herein, a monofunctional curable material contains one curable group or moiety, i.e., a functional group or moiety that is capable of polymerizing, entanglement, and / or crosslinking upon exposure to curing conditions (e.g., radiation, presence of calcium ions).

[0268] A multifunctional curable material contains two or more curable groups, for example, two, three, four or more curable groups. The multifunctional curable material can be, for example, a difunctional, trifunctional or tetrafunctional curable material containing two, three or four curable groups, respectively.

[0269] By "curable group" herein is meant a functional group that is capable of polymerizing and / or crosslinking upon exposure to appropriate curing conditions.

[0270] "Sclerotic collagen" refers to collagen (e.g., human recombinant collagen) as described herein in various embodiments, having one or more sclerotic groups as defined herein. According to some of the embodiments described herein, the sclerotic collagen is a multifunctional sclerotic material containing multiple sclerotic groups as defined herein.

[0271] The terms "sclerotic collagen," "sclerotic rh collagen," and "rh collagen having one or more (or at least one) sclerotic group" are used interchangeably herein.

[0272] According to some of the embodiments described herein, the hardenable collagen comprises an amino acid sequence as described herein in each embodiment and has one or more (preferably multiple) hardenable groups formed by the covalent attachment (directly or via a linker) of a compound containing a hardenable group to functional groups in the side chains of at least some of the amino acid residues that form the collagen, preferably to functional groups in the side chains of the amino acid residues. Alternatively, or in addition, hardenable groups can be created at the N-terminus and / or C-terminus of one or more of the units that form the collagen, for example, by covalently attaching (directly or via a linker) a compound containing a hardenable group to an amine or carboxylic acid.

[0273] According to some of the embodiments described herein, at least a portion of the curable groups of the curable collagen described herein are crosslinkable groups and crosslink upon exposure to curing conditions. Such curable collagen is also referred to herein as crosslinkable collagen, e.g., crosslinkable rh collagen (e.g., rh collagen produced by a plant described herein).

[0274] In some embodiments, the curable groups are capable of polymerizing and / or crosslinking by a free radical mechanism.

[0275] Examples of such curable groups include acrylic groups, such as acrylic acid groups, methacrylic acid groups, acrylamide groups, and methacrylamide groups. Other free radical curable groups can include thiols, vinyl ethers, and other groups characterized by a reactive double bond.

[0276] In some embodiments, the curable groups can be polymerized and / or crosslinked by other mechanisms, such as cationic polymerization or (cationic or anionic) ring-opening polymerization. Examples of such curable groups include, but are not limited to, epoxy-containing groups, caprolactam, caprolactone, oxetane, and vinyl ethers.

[0277] Other curable groups include, for example, the formation of an amide bond between a functional carboxylic acid and an amine group (each being a curable group that reacts with the other to crosslink), the formation of a urethane between an isocyanate group and a hydroxyl group by polycondensation in the presence of a catalyst and / or upon exposure to ultraviolet light, and the formation of a disulfide bond between two thiols.

[0278] Any other curable group is contemplated.

[0279] The generation of hardenable groups on hardenable collagen can be achieved by direct chemical reaction of a material containing or capable of generating hardenable groups with chemically compatible functional groups present on collagen, as described herein, or by means of spacers or linkers using chemistries well known in the art. For example, a material containing hardenable groups and functional groups can be reacted with compatible functional groups on collagen (e.g., functional groups on amino acid side chains) so that the hardenable groups become substituents on the amino acid side chains.

[0280] In some embodiments, compatible functional groups are first generated within the collagen by chemical modification of the collagen's inherent chemical groups, and then reacted with a material that contains or generates curable groups upon reaction.

[0281] When the hardenable collagen contains more than one hardenable group, the hardenable groups may be the same or different.

[0282] According to some of the embodiments described herein, at least some or all of the curable groups in the curable collagen of the present embodiments are photopolymerizable groups (e.g., ultraviolet curable groups) that can polymerize and / or crosslink upon exposure to radiation as described herein.

[0283] According to some of the embodiments described herein, the curable group is a photocurable or photopolymerizable group (eg, an acrylate or methacrylate).

[0284] The hardenable collagen described herein having acrylic (e.g., methacrylate) groups is also referred to herein as acrylated or methacrylated or (meth)acrylated collagen, e.g., (meth)acrylated rh collagen.

[0285] Alternatively, or in addition, the curable groups are thiol-containing groups, which upon curing result in disulfide crosslinks.

[0286] The sclerosing collagen described herein having thiol-containing groups is also referred to herein as thiolated collagen, eg, thiolated rh collagen.

[0287] Alternatively, or in addition, the hardenable groups or moieties are hardened by chemical reaction such as conjugation (using a coupling agent such as EDC) or glycation.

[0288] According to some embodiments, the curable groups include amine and carboxyl groups that form peptide bonds upon curing.

[0289] According to some of the embodiments described herein, at least some or all of the hardenable groups in the hardenable collagen of the present embodiments are acrylic groups (eg, methacrylic groups) as defined herein.

[0290] According to some of the embodiments described herein, acrylic groups such as methacrylamide can be generated by reacting an acrylate or methacrylate (e.g., acrylic acid, methacrylic acid, acrylic or methacrylic esters, acrylic or methacrylic anhydrides) with an amine functional group (e.g., of a lysine residue).

[0291] According to some embodiments of the present invention, the degree of hardening (e.g., degree of cross-linking) can be determined by the number of hardening groups in the hardenable collagen described herein, and the number can be manipulated to achieve a desired degree of hardening (e.g., degree of cross-linking).

[0292] The degree of cure affects the mechanical and / or physical and / or biological properties of the hardened material and can be manipulated to provide desired properties to the composite scaffold.

[0293] According to some of the embodiments described herein, the hardenable collagen has a plurality of acrylamide or methacrylamide hardenable groups formed by reaction with lysine residues as described herein.

[0294] According to some of the embodiments described herein, the hardenable collagen has a plurality of acrylamide or methacrylamide hardenable groups that substitute for the amine groups of lysine residues in the collagen.

[0295] In some embodiments, at least 50%, or at least 60%, or at least 70% of the lysine residues in the collagen are substituted with methacrylamide or acrylamide groups. In some embodiments, the hardenable collagen is characterized in that 70%-100%, or 80%-100%, or 90%-100% of its lysine residues (including intermediate values ​​and subranges therebetween) are substituted with methacrylamide or acrylamide groups.

[0296] According to some of the embodiments described herein, the modeling material formulation, upon solidification, forms a hydrogel material by crosslinking the rh collagen within the aqueous carrier (optionally with other hardenable components in the modeling formulation).

[0297] As used herein and in the art, the term "hydrogel" refers to a three-dimensional fibrous network containing at least 20%, usually at least 50%, or at least 80%, and up to about 99.99% water (by mass). Hydrogels can be considered materials that are mostly water but behave like solids or semi-solids due to a three-dimensional cross-linked solid-like network formed by polymer chains (e.g., collagen chains) within a liquid dispersion medium. The polymer chains are interconnected (cross-linked) by chemical bonds (covalent, hydrogen, and ionic / complex / metallic bonds, usually covalent).

[0298] Throughout this specification, when polymer chains or polymeric materials are mentioned, this includes polymeric biological materials (eg, macromolecules) such as peptides, proteins, oligonucleotides, and nucleic acids.

[0299] Hydrogels can take physical forms ranging from soft, brittle, and weak to hard, resilient, and strong. Soft hydrogels are characterized by rheological parameters, including elastic and viscoelastic parameters, while stiff hydrogels are properly characterized by tensile strength parameters, elastic modulus, storage modulus, and loss modulus, as these terms are known in the art.

[0300] The softness / hardness of a hydrogel is governed, among other things, by the chemical composition of the polymer chains, the "degree of cross-linking" (the number of interconnecting links between the chains), the content and composition of the aqueous medium, and the temperature.

[0301] According to some embodiments of the present invention, hydrogels may also include polymeric and / or fibrous elements that are not chemically bonded to the main crosslinked network, but rather are mechanically entangled and / or intertwined. Such polymeric fibrous elements may be woven (e.g., as in a mesh structure) or nonwoven, and in some embodiments, may serve as a reinforcing material for the hydrogel's fibrous network. Non-limiting examples of such polymers include polycaprolactone, gelatin, gelatin methacrylate, alginate, alginate methacrylate, chitosan, chitosan methacrylate, glycol chitosan, glycol chitosan methacrylate, hyaluronic acid (HA), HA methacrylate, and other non-crosslinked natural or synthetic polymer chains.

[0302] Alternatively, or in addition, such polymers may chemically bond to the main crosslinked network of the hydrogel, for example, by acting as crosslinkers or by forming part of the three-dimensional network of the hydrogel (e.g., when curable derivatives of such components are used).

[0303] In some embodiments, the hydrogel is porous, and in some embodiments, at least a portion of the pores in the hydrogel are nanopores, with an average volume in the nanoscale range.

[0304] According to some of the embodiments described herein, the rh collagen-containing modeling material formulation further comprises one or more additional materials, such as one or more additional hardenable materials, one or more non-hardenable materials, and / or one or more biological components or materials.

[0305] According to some of the embodiments described herein, the print medium (build material) includes one or more additional materials, such as one or more additional curable materials, one or more non-curable materials, and / or one or more biological components.

[0306] According to some of the embodiments described herein, additional materials are included in the rh collagen-containing formulation or one or more other modeling material formulations.

[0307] Additional curable materials that may be included in the rh collagen formulation or one or more other modeling material formulations may be any curable material as defined herein, and are preferably biocompatible materials.

[0308] In some embodiments, the additional hardenable material is or comprises a hydrogel, as defined herein, and is capable of forming a solidified modeling material upon exposure to curing conditions that cause crosslinking and / or copolymerization reactions, typically by further crosslinking and / or copolymerization. Such hardenable materials are also referred to herein as hydrogel hardenable materials.

[0309] In some embodiments described herein, the curable material is or comprises a hydrogel-forming material, as defined herein, that is capable of forming a hydrogel as a solidified modeling material, typically by crosslinking, entanglement, polymerization, and / or copolymerization, upon exposure to curing conditions that result in crosslinking, polymerization, and / or copolymerization reactions. Such curable materials are also referred to herein as hydrogel-forming curable materials or gel-forming materials.

[0310] According to embodiments of the present invention, the hydrogel may be of biological origin or synthetically prepared.

[0311] According to some embodiments of the present invention, the hydrogel is biocompatible and the activity of the biological moiety is maintained when the biological moiety is impregnated or deposited in the hydrogel, i.e., the activity of the biological moiety changes by no more than 30%, or no more than 20%, or no more than 10% compared to the activity of the biological moiety in a physiological medium.

[0312] Examples of polymers or copolymers that can be used to form hydrogels according to the present invention include polyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides, polyvinylpyrrolidones, and copolymers of any of the foregoing. Other examples include polyesters, polyurethanes, and poly(ethylene glycol), which can be functionalized with crosslinking groups or used in combination with compatible crosslinkers.

[0313] Some specific, non-limiting examples include poly(2-vinylpyridine), poly(acrylic acid), poly(methacrylic acid), poly(N-isopropylacrylamide), poly(N,N'-methylenebisacrylamide), poly(N-(N-propyl)acrylamide), poly(methacrylic acid), poly(2-hydroxyacrylamide), poly(ethylene glycol) acrylate, poly(ethylene glycol) methacrylate, and polysaccharides such as hyaluronic acid, dextran, alginate, agarose, and copolymers of any of the foregoing.

[0314] Hydrogel precursors (hydrogel-forming materials) that form such polymer chains (including any combination thereof) are contemplated.

[0315] Hydrogels are typically formed from or in the presence of di-, tri-, or polyfunctional monomers, oligomers, or polymers, collectively referred to as hydrogel precursors, or hydrogel formers, or hydrogel-forming materials, or simply crosslinkers as described herein, having two, three, or more polymerizable groups. The presence of more than one polymerizable group allows such precursors to be crosslinked, allowing for the formation of a three-dimensional network.

[0316] Examples of crosslinkable monomers include, but are not limited to, the family of diacrylate and triacrylate monomers having two or three polymerizable functional groups (one of which can be considered the crosslinkable functional group). Examples of diacrylate monomers include methylene diacrylate and poly(ethylene glycol). n Examples of triacrylate monomers include, but are not limited to, the family of dimethacrylates (nEGDMA) or diacrylates. Examples of triacrylate monomers include trimethylolpropane triacrylate, pentaerythritol triacrylate, tris(2-hydroxyethyl)isocyanurate triacrylate, isocyanuric acid tris(2-acryloyloxyethyl)ester, ethoxylated trimethylolpropane triacrylate, poly(ethylene glycol) n Examples include, but are not limited to, trimethacrylate or triacrylate, pentaerythrityl triacrylate and glycerol triacrylate, phosphinylidintris(oxyethylene)triacrylate.

[0317] In some of the embodiments described herein, the curable material, whether monomeric or oligomeric, can be a monofunctional curable material or a multifunctional curable material.

[0318] Examples of polymers or copolymers that can be used to form hydrogels according to the present invention include polyacrylates, polymethacrylates, polyacrylamides, polymethacrylamides, polyvinylpyrrolidones, and copolymers of any of the foregoing. Other examples include polyesters, polyurethanes, and poly(ethylene glycol), which can be functionalized with crosslinking groups or used in combination with compatible crosslinkers.

[0319] Some specific, non-limiting examples include poly(2-vinylpyridine), poly(acrylic acid), poly(methacrylic acid), poly(N-isopropylacrylamide), poly(N,N'-methylenebisacrylamide), poly(N-(N-propyl)acrylamide), poly(methacrylic acid), poly(2-hydroxyacrylamide), poly(ethylene glycol) acrylate, poly(ethylene glycol) methacrylate, poly(ethylene glycol) diacrylate, poly(ethylene glycol) dimethacrylate, and polysaccharides such as dextran, alginate, agarose, and copolymers of any of the foregoing.

[0320] Hydrogel precursors (hydrogel-forming materials) that form such polymer chains (including any combination thereof) are contemplated.

[0321] Curable materials that can be used in the field of bioprinting are mainly based on naturally occurring materials that can be isolated from animal or human tissues, such as Matrigel, alginate, pectin, xanthan gum, gelatin, chitosan, fibrin, cellulose and hyaluronic acid, or recombinantly produced or synthetically prepared materials, such as polyethylene glycol; PEG, gelatin methacrylate; GelMA, poly(propylene oxide); PPO, poly(ethylene oxide); PEO; PEG, polyethylene glycol. These include Fmoc-peptide-based hydrogels such as Fmoc-diacrylate, polyglutamic acid, gelatin methacrylate; GelMA, PLGA / PLLA, poly(dimethylsiloxane); nanocellulose; Pluronic F127, short dipeptides (FF), Fmoc-FF-OH, Fmoc-FRGD-OH, Fmoc-RGDF-OH, Fmoc-2-Nal-OH, Fmoc-FG-OH, and thermoplastic polymers such as polycaprolactone (PCL), polylactic acid (PLA), or poly(D,L-lactide-co-glycolide).

[0322] Examples of curable materials that can be used in the context of the present embodiments include, but are not limited to, Matrigel, gelatin methacrylate (GelMA), nanocellulose (UV-curable nanoscale structured materials such as cellulose nanocrystals (CNC), cellulose nanofibrils (CNF), and bacterial cellulose (BC), also known as microbial cellulose), Pluronic® materials such as Pluronic F127 and UV-curable Pluronic F127-diacrylate (DA), which are flowable at low temperatures and form gels at elevated temperatures above their critical micelle concentration (CMC), hyaluronic acid (HA), acrylated hyaluronic acid (AHA), methacrylated hyaluronic acid (MAHA), poly(ethylene glycol) diacrylate (PEGDA), alginate, xanthan gum, pectin, glutaraldehyde, chitosan that can be crosslinked with chemical agents such as genipin or sodium tripolyphosphate (TPP).

[0323] According to some of the embodiments described herein, the curable formulation further comprises a biocompatible synthetic material having a curable moiety, which upon curing (e.g., upon exposure to curing conditions described herein) forms a biocompatible synthetic polymer, also referred to herein as a curable biocompatible synthetic polymer or curable synthetic polymer.

[0324] According to some of the embodiments described herein, the curable formulation comprises a biocompatible synthetic polymer having one or more curable groups (e.g., polymerizable and / or crosslinkable groups) as described herein, i.e., a curable biocompatible synthetic polymer. Such materials, also referred to herein as modified biocompatible synthetic polymers, or modified derivatives thereof, or photopolymerizable modified derivatives thereof, or curable derivatives thereof, typically upon curing crosslink with themselves and / or other components in the formulation (e.g., curable rh collagen and / or crosslinking agents) to yield a crosslinked biocompatible synthetic polymer.

[0325] According to some of the embodiments described herein, the biocompatible synthetic polymer is or includes polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), poly(N-isopropylacrylamide) (PNIPAAm), poly-4-hydroxybutyrate (P4HB), or any copolymer thereof, or combination thereof.

[0326] According to some of the embodiments described herein, the curable formulation comprises precursors that, upon exposure to curing conditions, polymerize and / or crosslink to form the biocompatible synthetic polymers described above.

[0327] According to some embodiments described herein, the curable formulation comprises one or more of polylactic acid (PLA) with curable groups, polyglycolic acid (PGA) with curable groups, polycaprolactone (PCL) with curable groups, poly(lactic-co-glycolic acid) (PLGA) with curable groups, polyethylene glycol (PEG) with curable groups, polyvinyl alcohol (PVA) with curable groups, poly(N-isopropylacrylamide) (PNIPAAm), poly-4-hydroxybutyrate (P4HB) with curable groups, and copolymers of any of the foregoing with curable groups.

[0328] Each of the above polymers can have one or more curable groups.

[0329] Each of the above polymers can have one or more curable groups that form part of its original structure, or can be synthesized by attaching such groups directly or via a linker to one or more compatible functional groups of the polymer.

[0330] Synthetic polymers having one or more curable groups are also referred to interchangeably herein as modified synthetic polymers or modified derivatives thereof.

[0331] According to some embodiments described herein, the curable formulation comprises a modified PGA or a modified derivative thereof or a photopolymerizable modified derivative thereof, a modified PCL or a modified derivative thereof or a photopolymerizable modified derivative thereof, a modified PLGA or a modified derivative thereof or a photopolymerizable modified derivative thereof, a modified PEG or a modified derivative thereof or a photopolymerizable modified derivative thereof, a modified PVA or a modified derivative thereof or a photopolymerizable modified derivative thereof, a PNIPAAm or a modified derivative thereof, or any combination thereof. Modifications include, but are not limited to, (meth)acrylation and thiolation of biocompatible synthetic polymers. In some embodiments, the modified PEG comprises poly(ethylene glycol) diacrylate (PEGDA).

[0332] In some embodiments described herein, the curable formulations contain one or more multifunctional curable materials (monomers or polymeric materials) that, upon solidification, form biocompatible synthetic polymers or react with biocompatible synthetic polymers present in the formulation. Such materials act as crosslinkers, crosslinking agents, or crosslinking agents, and are also referred to herein as crosslinkers, crosslinking agents, or crosslinking agents. The crosslinking agents can interact with synthetic polymers (e.g., modified synthetic polymers) and / or the curable collagen described herein to form a crosslinked polymer network. An exemplary crosslinking agent includes ethoxylated (15) trimethylolpropane triacrylate, such as that commercially available under the trade name SR9035. Similar multifunctional (e.g., difunctional or trifunctional) crosslinkers are contemplated. In some embodiments, the crosslinking agent is a multifunctional PEG (e.g., PEG diacrylate or PEG triacrylate).

[0333] According to some of the embodiments described herein, the hardenable formulation comprises a biocompatible synthetic polymer as described herein and is capable of hardening, e.g., by crosslinking, upon exposure to curing conditions in the presence of other components in the formulation (e.g., a crosslinker and / or rh collagen as described herein).

[0334] According to some embodiments described herein, the curable formulation comprises a curable rh collagen as described herein in any and any combination of embodiments, and one or more biocompatible synthetic polymers independently having one or more curable groups as described herein in any and any combination of embodiments, hi some embodiments, the curable formulation further comprises a crosslinker as described herein.

[0335] The hardenable formulation also includes the hardenable rh collagen described herein in any of the embodiments and any combination thereof, and one or more biocompatible synthetic polymers described herein in any of the embodiments and any combination thereof that can be hardened, e.g., by crosslinking, and further includes a crosslinker described herein that crosslinks the polymer or crosslinks the polymer to one or more other components in the formulation (e.g., the hardenable rh collagen, other hardenable materials described herein, and / or other polymers) to facilitate hardening of the synthetic polymer.

[0336] According to some embodiments described herein, each of the biocompatible synthetic polymers having one or more curable groups independently has an average molecular weight (Mw or Mn) ranging from about 100 Daltons to about 100 kDa, including any intermediate values ​​and subranges therebetween. In some embodiments, the Mn of the synthetic polymer is from about 400 Da to about 100 kDa, or from about 400 Da to about 50 kDa, or from about 500 Da to about 50 kDa, or from about 100 Da to about 10 kDa, or from about 500 Da to about 10 kDa, or from about 1000 Da to about 10 kDa, or from about 100 Da to about 5 kDa, or from about 1000 Da to about 5 kDa, or from about 2 kDa to about 10 kDa, or from about 5 kDa to about 10 kDa, including any intermediate values ​​and subranges therebetween.

[0337] According to some of the embodiments described herein, the ratio of curable rh collagen (rh collagen having curable groups) to curable synthetic polymer (biocompatible synthetic polymer having curable groups and / or biocompatible synthetic polymer that can be crosslinked) in the curable formulation can be from about 10:1 to about 1:10 (including intermediate values ​​and subranges therebetween), such as from about 1:0.5 to about 1:2.0, or from about 5:1 to about 1:5, or from about 2:1 to 1:2, or from about 1:1 to 1:10, or from about 1:1 to 1:5, or from about 1:1 to 2:1, or from about 1:1 to 3:2, or from about 3:2 to 2:1.

[0338] In some embodiments, the ratio of curable rh collagen to curable biocompatible synthetic polymer is about 1:0.5, or 1:0.6, or 1:0.7, or 1:0.8, or 1:0.9, or 1:1, or 1:1.1, or 1:1.2, or 1:1.3, or 1:1.4, or 1:1. or 1:1.6, or 1:1.7, or 1:1.8, or 1:1.9, or 1:2.0.

[0339] According to some embodiments described herein, the hardenable formulation further comprises at least one extracellular matrix (ECM) component, such as an ECM protein (e.g., but not limited to, fibrinogen, collagen, fibronectin, vimentin, microtubule-associated protein 1D, neurite outgrowth factor (NOF), bacterial cellulose (BC), laminin, and gelatin). In some embodiments, the ECM component (in addition to rh collagen) comprises fibronectin, hyaluronic acid (HA), heparin, elastin, or laminin, or any combination thereof, as detailed herein.

[0340] In some of these embodiments, the ECM component comprises one or more curable groups as described herein (also referred to herein as a curable ECM component, or a modified ECM component, or a crosslinkable ECM component, or a polymerizable ECM component). The curable groups can be covalently attached to functional groups of the ECM component directly or via a linker.

[0341] For example, the formulation may further comprise one or more of HA, modified HA, or polymerizable modified derivatives thereof (eg, (meth)acrylated or thiolated HA).

[0342] In some embodiments, the curable ECM component comprises modified fibronectin (e.g., (meth)acrylated or thiolated fibronectin). In some embodiments, the curable ECM component comprises modified heparin (e.g., (meth)acrylated or thiolated heparin). In some embodiments, the curable ECM component comprises modified elastin (e.g., (meth)acrylated or thiolated elastin). In some embodiments, the curable ECM component comprises modified laminin (e.g., (meth)acrylated or thiolated laminin). Optionally, the formulation can include an ECM component capable of being cured by crosslinking, e.g., in the presence of a crosslinking agent, as described herein for any of the embodiments. Such curable ECM components may or may not have one or more curable groups as described herein.

[0343] According to some embodiments described herein, the curable modeling formulation further comprises an integrin-binding material, e.g., an RGD-containing material, such as a cyclic RGD-containing material. In some embodiments, the integrin-binding material, e.g., the RGD-containing material, is a curable material capable of polymerizing and / or crosslinking upon exposure to the curing conditions described herein. The curable RGD-containing material can have curable groups or can be used with a crosslinker and can be crosslinked in the presence of the crosslinker.

[0344] In some of these embodiments, the integrin-binding material is curable and has one or more curable moieties or groups.

[0345] Thus, in some embodiments, the integrin-binding material comprises at least one Arg-Gly-Asp (RGD) moiety, or peptidomimetic thereof, and can optionally further comprise other amino acids, amino acid derivatives, or other chemical groups (e.g., alkylene chains) and / or one or more curable groups as described herein in any of the embodiments and any combination thereof.

[0346] In some embodiments, the RGD-containing material is an oligopeptide, which can be a cyclic oligopeptide (including, for example, monocyclic, bicyclic, and tricyclic oligopeptides) or a linear oligopeptide, and can contain 1 to 10 amino acids in addition to the Arg-Gly-Asp amino acid sequence.

[0347] An exemplary oligopeptide is a cyclic peptide that is or includes c[Arg-Gly-Asp-Phe-Lys].

[0348] In some embodiments, the integrin-binding material comprises two or more Arg-Gly-Asp-containing moieties, which may be the same or different.

[0349] Examples of Arg-Gly-Asp-containing materials include, but are not limited to, c(RGDfk), RGD4C, and other RGD-containing cyclic peptides such as those described in Haubner et al. [J. Am. Chem. Soc. 1996, 118, 7881-7891] and Capello, et al. [J. Nucl. Med. 2004, 45(10), 1716-20], WO 97 / 06791, and U.S. Pat. No. 5,773,412.

[0350] In some embodiments, the RGD-containing material can include two or more -Arg-Gly-Asp- moieties, either linked to each other or separated by one or more amino acids or other spacers, as described herein.

[0351] In exemplary embodiments, the RGD-containing material contains one or more cysteine ​​residues, which in some exemplary embodiments is RGD4C. Such RGD-containing materials can crosslink with themselves and / or other thiolated curable components in the formulation by means of forming intermolecular disulfide bonds.

[0352] In some embodiments, any of the RGD-containing materials described herein comprise one or more curable groups or moieties, as described herein, attached to functional groups within amino acid side chains and / or terminal amines or carboxylates, as described herein, e.g., one or more (meth)acrylic groups.

[0353] Alternatively, the RGD-containing material may contain one or more thiol groups, such as the thiol groups of a cysteine ​​residue, making the material curable. If desired, the thiol groups can be attached to the RGD-containing material directly or via a linker.

[0354] Alternatively, or in addition, any of the RGD-containing materials described herein, whether or not they contain curable groups or moieties, can be attached to a polymeric material (preferably a biocompatible synthetic polymer as described in any of the embodiments herein), or a hydrogel-forming material, or an ECM component, as described in any of the embodiments herein. One or more RGD-containing moieties or materials can be attached to the polymer backbone of the material directly or via a linker, and can be attached to one or more of the terminal units and / or backbone units. The polymer can have curable groups as described herein or can be crosslinked in the presence of a crosslinking agent. Alternatively, or in addition, the RGD-containing moiety has one or more curable groups (e.g., thiol and / or (meth)acrylic groups) as described herein.

[0355] Exemplary RGD-containing materials include one or more RGD-containing sequences described herein covalently attached to PEG, e.g., PEG-DA or PEG-TA, as described in any of the embodiments herein. According to some of the embodiments described herein, the hardenable moieties or groups of the rh collagen and the hardenable moieties or groups of the synthetic polymer are hardenable when subjected to the same hardening conditions. In some of these embodiments, the hardening conditions include irradiation (illumination), and such hardenable moieties or groups are photohardenable (photopolymerizable) moieties or groups.

[0356] According to some of the embodiments described herein, the curable moieties or groups of the rh collagen and synthetic polymer, and the curable moieties or groups of the ECM component (if present) and integrin-binding material (if present), are curable when subjected to the same curing conditions. In some of these embodiments, the curing conditions include irradiation (illumination), and such curable moieties or groups are photocurable (photopolymerizable) moieties or groups.

[0357] According to some of the embodiments described herein, the modeling material formulation is characterized by a viscosity of 2000 centipoise or less, or 1500 centipoise or less, at zero shear rate and 37°C, as determined using a rheometer (e.g., a Brookfield rheometer) according to methods well known in the art.

[0358] According to some of the embodiments described herein, the modeling material formulation is characterized by a viscosity of 2000 centipoise or less, or 1500 centipoise or less, at a shear rate of 5 L / sec and room temperature, as determined using a rheometer described herein.

[0359] In some of the embodiments described herein, the concentration of the curable recombinant human collagen in the modeling material formulation is in the range of 0.5 mg / mL to 50 mg / mL, or 0.5 mg / mL to 20 mg / mL, or 1 mg / mL to 50 mg / mL, or 1 mg / mL to 50 mg / mL, or 1 mg / mL to 40 mg / mL, or 1 mg / mL to 30 mg / mL, or 2 mg / mL to 20 mg / mL, or 5 mg / mL to 15 mg / mL, or 1 mg / mL to 10 mg / mL (including any intermediate values ​​and subranges therebetween).

[0360] In some of the embodiments described herein, the total concentration of the curable biocompatible synthetic polymer in the modeling material formulation is in the range of 1 mg / mL to 500 mg / mL, or 10 mg / mL to 500 mg / mL, or 1 mg / mL to 100 mg / mL, or 10 mg / mL to 100 mg / mL, or 50 mg / mL to 500 mg / mL, or 50 mg / mL to 300 mg / mL, or 1 mg / mL to 20 mg / mL, or 5 mg / mL to 50 mg / mL (including any intermediate values ​​or subranges therebetween).

[0361] In some of the embodiments described herein, the total concentration of the curable ECM component (if present) in the modeling material formulation is in the range of 0.01 mg / mL to 10 mg / mL, or 0.01 mg / mL to 5 mg / mL, or 0.01 mg / mL to 1 mg / mL, or 0.05 mg / mL to 1 mg / mL (including any intermediate values ​​and subranges therebetween).

[0362] In some of the embodiments described herein, the total concentration of the curable integrin-binding material (if present) in the modeling material formulation is stoichiometric relative to the curable rh collagen, and can be, for example, in the range of 1-50 μM, or 1-40 μM, or 1-30 μM, including any intermediate values ​​and subranges therebetween.

[0363] According to some of the embodiments described herein, the concentration of the curable recombinant human collagen in the modeling material formulation is in the range of 0.01-10 wt %, or 0.01-5 wt %, or 0.05-10 wt %, or 0.05-5 wt %, or 0.1-2 wt %, or 0.1-1 wt %, or 10-50 wt %, or 10-40 wt %, or 10-30 wt %, or 10-20 wt %, or 20-40 wt %, or 30-40 wt % by weight of the total weight of the formulation, including any intermediate values ​​and subranges therebetween.

[0364] According to some of the embodiments described herein, the concentration of the curable biocompatible synthetic polymer (or its precursor curable material) in the modeling material formulation is in the range of 0.1-10 wt %, or 0.1-5 wt %, or 0.1-2 wt %, or 0.5-2 wt %, or 1-60 wt %, or 1-50 wt %, or 10-60 wt %, or 5-50 wt %, or 10-40 wt %, or 5-30 wt %, or 10-20 wt %, or 5-15 wt %, including any intermediate values ​​or subranges therebetween, of the total weight of the components of the formulation (not including the carrier, as described below).

[0365] According to some of the embodiments described herein, the concentration of the ECM component having a curable group as described in any of the embodiments herein is in the range of 0.001 to 0.1 wt %, including any intermediate values ​​and subranges therebetween, of the total weight of the components of the formulation (not including the carrier as described below).

[0366] According to some of the embodiments described herein, the curable (modeling, bioink) formulation comprises: a curable rh collagen as described herein in any of the embodiments and any combination thereof, for example, a (e.g., plant-derived) rh collagen as described herein having a plurality of (meth)acrylic groups as described herein; a curable biocompatible synthetic polymer as described in any one of the embodiments and any combination thereof herein, for example, one or more of polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), poly(N-isopropylacrylamide) (PNIPAAm), poly-4-hydroxybutyrate (P4HB), or a copolymer of any of them, having one or more curable groups (e.g., (meth)acrylic and / or thiol) groups, as described in any one of the embodiments and any combination thereof herein; As described in any of the embodiments and any combination thereof herein, the curable material comprises an ECM component (e.g., one or more of hyaluronic acid, fibronectin, heparin, elastin, or laminin) having one or more curable groups (e.g., (meth)acrylic or thiol) groups, as described in any of the embodiments herein, and / or a curable ECM component used in combination with a crosslinker as described herein, and a curable RGD-containing material as described in any of the embodiments herein.

[0367] In some of these embodiments, the formulation further comprises an aqueous carrier (e.g., water optionally mixed with a water-soluble organic acid), the concentration of the curable rh collagen is in the range of 1-10 mg / mL, the concentration of the curable synthetic polymer is in the range of 50-300 mg / mL, the concentration of the ECM component is in the range of 0-1 mg / mL, and the amount of RGD-containing material is stoichiometrically adjusted to the rh collagen.

[0368] Exemplary formulations are described in the Examples section below.

[0369] Exemplary formulations according to some embodiments of the present invention include 0.1% to 1% by weight of the curable rh collagen and 5-15% by weight of the curable synthetic polymer, as described herein, based on the total weight of the formulation.

[0370] An exemplary formulation according to some embodiments of the present invention comprises 0.1-0.5 wt. % of the curable rh collagen and 10-15 wt. % of the curable synthetic polymer, as described herein, based on the total weight of the formulation.

[0371] The curable biocompatible synthetic polymer in these exemplary formulations can be, for example, a multifunctional (e.g., bifunctional) PEG-acrylate (e.g., PEG-DA), and as described herein, can have an average Mn value of 1 or greater. For example, one, two, three, or more PEG-DAs can be included, each characterized by a different Mn.

[0372] The concentration of curable rh collagen in a modeling material formulation can affect the rheological properties of the formulation and the resulting solidified formulation upon dispensing, and can be manipulated depending on the AM method and conditions used and the desired properties of the final object or portion thereof. Similarly, the concentration and type (e.g., amount of polymerizable groups, average Mn) of the curable biocompatible synthetic polymer (or its curable precursor) can affect the rheological properties of the formulation and the resulting solidified formulation upon dispensing, and can be manipulated depending on the AM method and conditions used and the desired properties of the final object or portion thereof.

[0373] According to this embodiment, the build material comprises at least one modeling material formulation comprising curable recombinant human collagen, as described in any of the embodiments herein. Such modeling material formulations are also referred to herein as rh collagen-containing formulations.

[0374] According to some of the embodiments described herein, the curable formulation further comprises a carrier, and in some of these embodiments, the carrier is an aqueous carrier.

[0375] The aqueous carrier can be water, a buffer solution characterized by a pH in the range of about 4 to about 10, or about 6 to about 8, or about 7 to about 7.4, a basic aqueous solution, or an acidic aqueous solution.

[0376] Aqueous carriers can contain salts and other water-soluble materials at various concentrations, hi some embodiments, the concentration of salt in the carrier is 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 any intermediate values ​​and subranges therebetween.

[0377] In some embodiments, the aqueous carrier contains a physiologically acceptable concentration of salt, such that the formulation is characterized by an osmolality near physiological osmolality.

[0378] In some embodiments, the aqueous carrier comprises a phosphate salt, such as monobasic sodium phosphate (NaHPO) and / or dibasic sodium phosphate (sodium hydrogen phosphate, NaHPO). In some embodiments, the total concentration of the phosphate salt is about 0.1 M.

[0379] In some embodiments, the aqueous carrier comprises NaCl or other physiologically acceptable salts.

[0380] In some embodiments, the aqueous carrier comprises a phosphate buffer, and in some embodiments, the aqueous carrier comprises a phosphate buffered saline solution comprising monobasic sodium phosphate and / or dibasic sodium phosphate and NaCl.

[0381] The phosphate buffered saline (PBS) can be commercially available PBS (e.g., DPBS) or a custom buffer characterized by a desired pH and / or osmolality.

[0382] In an exemplary embodiment, the aqueous carrier comprises a phosphate buffer containing a sodium phosphate salt described herein at a concentration of about 0.1 M and NaCl at a concentration of about 0 mM to about 200 mM, including intermediate values ​​and subranges therebetween.

[0383] Any other buffer solution can be used in the context of this embodiment.

[0384] In some of the embodiments described herein, the aqueous carrier comprises an acid.

[0385] In some embodiments, the concentration of the acid is less than 100 mM, for example, 0.1 mM to 50 mM, or 0.1 mM to 30 mM, or 0.1 mM to 40 mM, or 0.1 mM to 30 mM, or 1 to 30 mM, or 10 to 30 mM (including intermediate values ​​and subranges therebetween).

[0386] In some embodiments, the concentration of the acid is in the range of 0.01 to 0.1 wt %, or 0.01 to 0.05 wt %, including any intermediate values ​​and subranges therebetween.

[0387] The acid can be an inorganic acid (eg, HCl) or an organic acid (preferably water soluble at the concentrations described above, such as acetic acid or picric acid).

[0388] Non-setting materials other than the biological materials described herein can also be included in one or more of the modeling formulations described herein, for example, materials that impart specific properties to the formulation or to the solidified formulation and composite scaffold formed therefrom. Such properties can be physical properties (e.g., optical properties such as transparency or opacity, color, spectral properties, heat resistance, electrical properties, etc.) or mechanical or rheological properties such as viscosity, elasticity, storage modulus, loss modulus, stiffness, hardness, etc.

[0389] Examples of non-curable materials include thixotropic agents, reinforcing agents, toughening agents, fillers, colorants, pigments, dyes, and the like.

[0390] According to some of the embodiments described herein, the formulation is characterized by a neutral pH (eg, about 6 to about 8).

[0391] According to some of the embodiments described herein, the formulation is essentially characterized by the viscosity parameters described herein.

[0392] In embodiments using two or more modeling material formulations, the two or more formulations are rh collagen-containing formulations described herein that differ from one another in the presence, type, and / or concentration of additional materials included therein. For example, one formulation can include curable rh collagen, while another formulation can include curable rh collagen and one or more additional curable materials described herein (e.g., curable synthetic polymers). For example, one formulation can include curable rh collagen and an additional curable material, while another formulation can include curable rh collagen and another additional curable material described herein. For example, one formulation can include curable rh collagen and an additional curable material, while another formulation can include curable rh collagen and a non-curable material described herein. Any other combinations are contemplated.

[0393] In some of the embodiments described herein, all of the curable materials in the build material are cured under the same curing conditions, hi some embodiments, all of the curable materials are photocurable.

[0394] In some of the embodiments described herein, modeling material formulations that include a curable material further include an agent that promotes the hardening or solidification of the curable material upon exposure to curing conditions.

[0395] The concentration of the agent can be determined depending on the concentration of the curable material and the desired degree of cure (eg, the desired degree of crosslinking).

[0396] When the curable material is a photocurable material, the agent is a photoinitiator. The photoinitiator is selected depending on the curing mechanism (e.g., free radical, cationic, etc.).

[0397] A free radical photoinitiator can be any compound that generates free radicals upon exposure to radiation, such as ultraviolet or visible light, thereby initiating a polymerization reaction. Non-limiting examples of suitable photoinitiators include benzophenones (aromatic ketones) such as benzophenone, methylbenzophenone, Michler's ketone, and xanthone; acylphosphine oxide photoinitiators such as 2,4,6-trimethylbenzolidiphenylphosphine oxide (TMPO), 2,4,6-trimethylbenzoylethoxyphenylphosphine oxide (TEPO), lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and bisacylphosphine oxide (BAPO); benzoins and benzoin alkyl ethers such as benzoin, benzoin methyl ether, and benzoin isopropyl ether; and the like.

[0398] Examples of photoinitiators include, but are not limited to, the Irgacure® family, riboflavin, rose bengal, and the like.

[0399] Free radical photoinitiators can be used alone or in combination with coinitiators. Coinitiators are used with initiators that require a second molecule to generate active radicals in photocurable free radical systems. Benzophenone is an example of a photoinitiator that requires a second molecule, such as an amine, to generate free radicals. After absorbing radiation, benzophenone reacts with tertiary amines by hydrogen abstraction to produce α-amino radicals that initiate the polymerization of acrylates. Non-limiting examples of types of coinitiators include alkanolamines such as triethylamine, methyldiethanolamine, and triethanolamine.

[0400] Suitable cationic photoinitiators include, for example, compounds that form aprotic acids or Bronsted acids upon exposure to sufficient ultraviolet and / or visible light to initiate polymerization. The photoinitiator used can be a single compound, a mixture of two or more active compounds, or a combination of two or more different compounds, i.e., coinitiators. Non-limiting examples of suitable cationic photoinitiators include aryl diazonium salts, diaryliodonium salts, triarylsulfonium salts, triarylselenonium salts, and the like. One example of a cationic photoinitiator is a mixture of triarylsulfonium hexafluoroantimonate salts.

[0401] Non-limiting examples of suitable cationic photoinitiators include p-(octyloxyphenyl)phenyliodonium hexafluoroantimonate UVACURE 1600 (available from Cytec Company, USA), iodonium (4-methylphenyl)(4-(2-methylpropyl)phenyl)-hexafluorophosphate known as Irgacure 250 or Irgacure 270 (available from Ciba Specialty Chemicals, Switzerland), mixed arylsulfonium hexafluoroantimonate salts known as UVI 6976 and 6992 (available from Lambson Fine Chemicals, UK), diaryliodonium hexafluoroantimonate known as PC2506 (available from Polyset Company, USA), (tolylcumyl)iodonium tetrakis(pentafluorophenyl)borate known as Rhodorsil® Photoinitiator 2074 (available from Bluestar Silicones, USA), Tego Iodonium bis(4-dodecylphenyl)-(OC-6-11)-hexafluoroantimonate, known as PC1466 (available from Evonik Industries AG, Germany), is an example.

[0402] The concentration of the photoinitiator in the curable formulation can range from 0.1 to 3 wt%, or 0.1 to 2 wt%, or 0.5 to 2.5 wt%, or 0.1 to 2 wt%, or 0.5 to 1.5 wt% of the total weight of the formulation.

[0403] Chemical composition of the composite scaffold: In some embodiments, the 3D bioprinted composite scaffold comprises recombinant human collagen (rhcollagen) and a biocompatible synthetic polymer.

[0404] Those skilled in the art will understand that the terms "composite" or "composite scaffold" can encompass scaffolds comprising a combination of materials. In some embodiments, a composite scaffold is degradable. In some embodiments, a composite scaffold is biodegradable. In some embodiments, a composite scaffold is biocompatible. In some embodiments, a composite scaffold is partially degradable. In some embodiments, a composite scaffold is a mechanically degradable scaffold. In some embodiments, a composite scaffold comprises a biodegradable component and a mechanically degradable component. Those skilled in the art will understand that the biocompatible component of a composite scaffold is gradually reabsorbed or metabolized by the body after mechanically breaking down into smaller pieces over a period of time.

[0405] In some embodiments, the composite scaffold comprises collagen. In some embodiments, the composite scaffold comprises rh collagen. In some embodiments, the composite scaffold comprises collagen as described in any of the embodiments herein. In some embodiments, the collagen comprises human collagen. In some embodiments, the collagen comprises rh collagen. In some embodiments, the collagen comprises plant-derived collagen. In some embodiments, the collagen comprises plant-derived rh collagen. In some embodiments, the collagen comprises cross-linked collagen and / or hardenable rh collagen as described herein that has undergone cross-linking. In some embodiments, the collagen comprises cross-linked plant-derived recombinant human collagen, as detailed herein.

[0406] In some embodiments, the composite scaffold comprises a biocompatible synthetic polymer, such as a crosslinked biocompatible synthetic polymer and / or a curable biocompatible synthetic polymer described herein that has undergone polymerization and / or crosslinking.

[0407] Those skilled in the art will appreciate that the term "biocompatible" can encompass materials that are compatible with living tissues or organs without eliciting toxicity, immune response, damage, and the like.

[0408] Those skilled in the art will understand that the term "synthetic polymer" can include polymers that do not occur in nature and / or are not derived from naturally occurring materials, but are synthesized by chemical synthesis.

[0409] According to some of the embodiments described herein, the chemical composition of the composite scaffold is the result of subjecting the curable formulation described herein in any of the embodiments and any combination thereof to curing conditions.

[0410] According to some of the embodiments described herein, all of the curable groups or moieties in the components of the curable formulation are photocurable (e.g., photocurable, photopolymerizable, UV-curable), and the chemical composition of the composite scaffold is the result of subjecting the curable formulation described herein in any of the embodiments and any combination thereof to irradiation (e.g., ultraviolet-visible irradiation).

[0411] Without being bound by any particular theory, it is believed that when forming a composite scaffold as described herein in any of the embodiments, the curable materials crosslink to each other upon exposure to curing conditions to provide the hydrogel network described herein.

[0412] Those skilled in the art will understand that the terms "crosslink," "crosslinking," or "crosslinkable" refer to the attachment of at least two molecules to one another through chemical interactions (e.g., covalent bond formation, hydrogen bond formation, hydrophobic interactions, hydrophilic interactions, ionic interactions, or electrostatic interactions). Those skilled in the art will further understand that there are various methods of crosslinking. In some embodiments, such methods use photoreactive molecules and photoinduced reactions, for example, by illumination with infrared, ultraviolet, or white light sources, to generate crosslinked molecules. Those skilled in the art will understand that crosslinking is used to increase the stability and strength of matrices and scaffolds containing the crosslinking molecules described herein, but does not impair the properties of the matrix or scaffold as the matrix or scaffold is degraded, resorbed, or metabolized in the body over a period of time.

[0413] According to some of the embodiments described herein, upon exposure to curing conditions (e.g., irradiation), the curable materials undergo crosslinking and / or polymerization with each other (with the same curable component) and / or with each other to form a network of crosslinked materials (optionally in the form of a hydrogel as described herein).

[0414] In some embodiments, the composite scaffold comprises cross-linked plant-derived human collagen and two different synthetic polymers (e.g., cross-linked polymers), three different synthetic polymers, four different synthetic polymers, five different synthetic polymers, six different synthetic polymers, or seven different synthetic polymers. In some embodiments, the composite scaffold comprises at least two different synthetic polymers, at least three different synthetic polymers, at least four different synthetic polymers, at least five different synthetic polymers, at least six different synthetic polymers, or at least seven different synthetic polymers. Two or more of the cross-linked rh collagen and cross-linked polymers can be cross-linked to each other to form a cross-linked network as described herein.

[0415] The ranges and specific ratios of rh collagen to biocompatible synthetic polymer are as described above and follow the ratios described herein for the curable formulations of any of the embodiments.

[0416] In some embodiments, the composite scaffold comprises a cross-linked biocompatible synthetic polymer, hi some embodiments, the cross-linked biocompatible synthetic polymer comprises cross-linked PLA, cross-linked PGA, cross-linked PCL, cross-linked PLGA, cross-linked PEG, cross-linked PEGDA, cross-linked PVA, cross-linked poly-4-hydroxybutyrate (P4HB), cross-linked PNIPAAm, or any combination thereof.

[0417] In some embodiments, the composite scaffold comprises (crosslinked) rh collagen, a (e.g., crosslinked) synthetic polymer, and at least one extracellular matrix (ECM) component, e.g., a crosslinked ECM component. In some embodiments, the ECM component, in addition to rh collagen, comprises fibronectin, HA, heparin, elastin, or laminin, or any combination thereof, as detailed herein. In some embodiments, the HA comprises crosslinked HA. In some embodiments, the heparin comprises crosslinked heparin. In some embodiments, the elastin comprises crosslinked HA. In some embodiments, the laminin comprises crosslinked laminin.

[0418] In some embodiments, the composite scaffold comprises two different ECM components, three different ECM components, four different ECM components, or five different ECM components. In some embodiments, the composite scaffold comprises at least two different ECM components, at least three different ECM components, at least four different ECM components, or at least five different ECM components. The rh collagen, one or more biocompatible synthetic polymers, and one or more ECM components can be crosslinked such that at least two, at least three, or all of these materials are crosslinked to each other to form the networks described herein.

[0419] In some embodiments, the collagen comprises crosslinked collagen. In some embodiments, the rh collagen comprises crosslinked rh collagen. In some embodiments, the curable (e.g., (meth)acrylated) rh collagen crosslinks only to itself under curing conditions (e.g., irradiation / illumination). In some embodiments, the curable (e.g., (meth)acrylated) rh collagen crosslinks to other curable rh collagens (e.g., thiolated rh collagen) under curing conditions (e.g., irradiation / illumination). In some embodiments, the curable (e.g., (meth)acrylated and / or thiolated) rh collagen crosslinks to other curable materials (e.g., (meth)acrylated / thiolated curable components) in the curable formulation under curing conditions, as described in any of the embodiments herein. In some embodiments, the curable (e.g., (meth)acrylated) rh collagen crosslinks to curable (e.g., (meth)acrylated) HA under curing conditions (e.g., irradiation / illumination). In some embodiments, curable (e.g., thiolated) rh collagen crosslinks to thiolated HA under curing conditions (e.g., irradiation / illumination). In some embodiments, curable (e.g., (meth)acrylated) rh collagen crosslinks to curable (e.g., (meth)acrylated) PVA under curing conditions (e.g., irradiation / illumination). In some embodiments, curable (e.g., thiolated) rh collagen crosslinks to curable (e.g., thiolated) PVA under curing conditions (e.g., irradiation / illumination). In some embodiments, curable (e.g., (meth)acrylated) rh collagen crosslinks to curable (e.g., (meth)acrylated) PEG under curing conditions (e.g., irradiation / illumination). In some embodiments, curable (e.g., thiolated) rh collagen crosslinks to curable (e.g., thiolated) PEG under curing conditions (e.g., irradiation / illumination).

[0420] In some embodiments, (meth)acrylated rh collagen crosslinks to PEG-DA under curing conditions (e.g., irradiation / illumination). In some embodiments, thiolated rh collagen crosslinks to PEG-DA under curing conditions (e.g., irradiation / illumination). In some embodiments, (meth)acrylated rh collagen crosslinks to methacrylated or thiolated PLA under curing conditions (e.g., irradiation / illumination). In some embodiments, thiolated rh collagen crosslinks to methacrylated or thiolated PLA under curing conditions (e.g., irradiation / illumination). In some embodiments, methacrylated rh collagen crosslinks to methacrylated or thiolated PGA under curing conditions (e.g., irradiation / illumination). In some embodiments, thiolated rh collagen crosslinks to methacrylated or thiolated PGA under curing conditions (e.g., irradiation / illumination). In some embodiments, methacrylated rh collagen crosslinks to methacrylated or thiolated PCL under curing conditions (e.g., irradiation / illumination). In some embodiments, thiolated rh collagen crosslinks to methacrylated or thiolated PCL under curing conditions (e.g., irradiation / illumination). In some embodiments, methacrylated rh collagen crosslinks to methacrylated or thiolated PLGA under curing conditions (e.g., irradiation / illumination). In some embodiments, thiolated rh collagen crosslinks to methacrylated or thiolated PLGA under curing conditions (e.g., irradiation / illumination). In some embodiments, methacrylated rh collagen crosslinks to methacrylated or thiolated PNIPAAm under curing conditions (e.g., irradiation / illumination). In some embodiments, thiolated rh collagen crosslinks to methacrylated or thiolated PNIPAAm under curing conditions (e.g., irradiation / illumination).

[0421] In some embodiments, methacrylated rh collagen crosslinks to methacrylated OC under curing conditions (e.g., irradiation / illumination). In some embodiments, methacrylated rh collagen crosslinks to thiolated OC under curing conditions (e.g., irradiation / illumination). In some embodiments, methacrylated rh collagen crosslinks to N-(2-hydroxyethyl)acrylamide (HEAA) under curing conditions (e.g., irradiation / illumination). In some embodiments, thiolated rh collagen crosslinks to N-(2-hydroxyethyl)acrylamide (HEAA) under curing conditions (e.g., irradiation / illumination).

[0422] Degradable implants: According to an aspect of some embodiments of the present invention there is provided a soft tissue implant comprising a composite scaffold as described herein in any of the embodiments and any combination thereof.

[0423] Those skilled in the art will understand that the terms "implant" and "implantable" can encompass any material that can be fully or partially, permanently or temporarily implanted, inserted, embedded and / or transplanted into the body of a subject (e.g., an animal or human, preferably a mammal), e.g., attached to tissue, muscle, organ or other part of the subject's body.

[0424] According to some embodiments, the soft tissue implant is a biocompatible, degradable implant, as those terms are defined throughout this specification.

[0425] Those skilled in the art will understand that the term "degradable," which is interchangeable with the term "resorbable," can encompass the ability to be broken down or metabolized within a subject over a period of time when implanted within a subject. In some embodiments, the implants described herein include biodegradable implants. In some embodiments, the implants described herein include mechanically degraded implants. In some embodiments, the implants described herein include both biodegradable and mechanically degradable implants. In some embodiments, the implants described herein include naturally degradable implants.

[0426] According to some embodiments, an "implant" is a medical device manufactured to replace lost biological structures, support damaged biological structures, and / or augment existing biological structures. In some embodiments, the implant according to this embodiment is an implant for the reconstruction of body tissue and / or the restoration of tissue or organ function.

[0427] Examples of implants include, but are not limited to, breast implants for breast reconstruction or augmentation after mastectomy, salivary gland implants for salivary gland function reconstruction, and pancreatic implants for restoration of pancreatic islet function (i.e., insulin and / or glucagon secretion). Further examples are described below. According to some embodiments, the soft tissue implant is a biocompatible, degradable implant, as these terms are defined throughout this specification.

[0428] In some embodiments, the implants described herein can gradually degrade over a period of about 1 to 36 months. In some embodiments, the implant degrades within a period of about 3 to 36 months. In some embodiments, the implant degrades within a period of about 1 to 24 months. In some embodiments, the implant degrades within a period of about 1 to 12 months. In some embodiments, the implant degrades within a period of about 12 to 24 months. In some embodiments, the implant degrades within a period of about 24 to 36 months. In some embodiments, the implant degrades within about 1 month. In some embodiments, the implant degrades within about 3 months. In some embodiments, the implant degrades within about 6 months. In some embodiments, the implant degrades within about 12 months. In some embodiments, the implant degrades within about 24 months. In some embodiments, the implant degrades within about 36 months. In some embodiments, the implant degrades within less than 1 month. In some embodiments, the implant degrades within less than 3 months. In some embodiments, the implant degrades within less than 6 months. In some embodiments, the implant degrades within less than 12 months. In some embodiments, the implant degrades in less than 24 months, hi some embodiments, the implant degrades in less than 36 months.

[0429] In some embodiments, implants comprising the composite scaffolds described herein degrade over time after implantation into a subject. In some embodiments, implants comprising the composite scaffolds are bioresorbable. In some embodiments, implants described herein are designed to degrade or disintegrate over time, with the scaffold being replaced by newly formed tissue. In some embodiments, the implants comprise biodegradable or naturally dissolving implants. In some embodiments, implants comprising the composite scaffolds are made from materials that dissolve or are absorbed by the body. In some embodiments, the implants degrade over a period of time. In some embodiments, the degradation includes gradual degradation over 1 month, 3 months, 6 months, 12 months, 24 months, or 36 months.

[0430] In some embodiments, the 3D bioprinted degradable implant degrades up to 36 months after implantation. In some embodiments, the 3D bioprinted degradable implant degrades up to 24 months after implantation. In some embodiments, the 3D bioprinted degradable implant degrades up to 12 months after implantation. In some embodiments, the 3D bioprinted degradable implant degrades between 24 and 36 months after implantation. In some embodiments, the 3D bioprinted degradable implant degrades between 12 and 24 months after implantation. In some embodiments, the 3D bioprinted degradable implant degrades between 6 and 18 months after implantation. In some embodiments, the 3D bioprinted degradable implant degrades between 3 and 12 months after implantation. In some embodiments, the 3D bioprinted degradable implant degrades between 1 and 12 months after implantation. In some embodiments, the 3D bioprinted degradable implant is within about 3 months of implantation, within about 6 months of implantation, within about 7 months of implantation, within about 8 months of implantation, within about 9 months of implantation, within about 10 months of implantation, within about 11 months of implantation, within about 12 months of implantation, within about 13 months of implantation, within about 14 months of implantation, within about 15 months of implantation, within about 16 months of implantation, within about 17 months of implantation, within about 18 months of implantation, within about 19 months of implantation, within about 20 months of implantation, within about 21 months of implantation, within about 22 months of implantation, within about 23 months of implantation, within about 24 months of implantation, within about 25 months of implantation, within about 26 months of implantation, within about 27 months of implantation, within about 28 months of implantation, within about 29 months of implantation, within about 30 months of implantation, within about 31 months of implantation, within about 32 months of implantation, within about 33 months of implantation, within about 34 months of implantation, within about 35 months of implantation, within about 36 months of implantation, within about 37 months of implantation, within about 38 months of implantation, within about 39 months of implantation, within about 40 months of implantation, within about 41 months of implantation, within about 42 months of implantation, within about 43 months of implantation, within about 44 months of implantation, within about 45 months of implantation, within about 46 months of implantation, within about 47 months of implantation, within about 48 months of implantation, within about 49 months of implantation, within about 50 months of implantation, within about 51 months of implantation, within about 52 months of implantation, within about 53 months of implantation, within about 54 months of implantation, within about 20 months after transplantation, within about 21 months after transplantation, within about 22 months after transplantation, within about 23 months after transplantation, within about 24 months after transplantation, within about 25 months after transplantation, within about 26 months after transplantation, within about 27 months after transplantation, within about 28 months after transplantation, within about 29 months after transplantation, within about 30 months after transplantation, within about 31 months after transplantation, within about 32 months after transplantation, or within about 33 months after transplantation, within about 34 months after transplantation, within about 35 months after transplantation, or within about 36 months after transplantation.

[0431] In some embodiments, the 3D bioprinted degradable implants disclosed herein can be implanted in a subject in need thereof. In some embodiments, the 3D bioprinted degradable implants are used to replace or reconstruct breast tissue. In some embodiments, the 3D bioprinted degradable implants are used in subjects undergoing mastectomy requiring breast reconstruction. In some embodiments, the 3D bioprinted degradable implants are used in cosmetic, plastic, and reconstructive surgical procedures resulting from surgery, disease, or trauma. In some embodiments, the 3D bioprinted degradable implants gradually degrade over time. In some of these embodiments, the 3D bioprinted degradable implants are replaced with newly formed tissue.

[0432] According to some of the embodiments described herein, the soft tissue implant is a breast implant.

[0433] Those skilled in the art will understand that the term "breast implant" can encompass implants that are inserted beneath or within the breast tissue or beneath the pectoral muscle for breast augmentation, reconstruction, or replacement.

[0434] According to some of the embodiments described herein, the soft tissue is facial (e.g., nose, ear, chin, cheek, eye, lip, etc.) tissue, neck tissue, muscle tissue, joint tissue, jaw tissue, hip tissue, hand tissue, breast tissue, brain tissue, liver tissue, cartilage, connective tissue, heart tissue, lung tissue, or gonadal tissue, and the soft tissue implant, as described herein, is for implantation into or near one or more of these tissues, and / or one or more oral cavities comprising one or more of these tissues, and / or one or more body organs comprising one or more of these tissues.

[0435] According to some embodiments described herein, the soft tissue implant is a salivary gland implant, a pancreatic implant, a bone implant, an implant for reconstructing anterior cruciate ligament rupture, a craniofacial reconstruction implant, a maxillofacial reconstruction implant, a complex jaw surgery implant, a reconstruction implant after tumor resection, an implant for tissue reconstruction after melanoma resection, an implant for tissue reconstruction after head and neck cancer resection, an ear implant, a nose implant, a chest wall reconstruction implant, an orthopedic implant, a cartilage reconstruction implant, and a delayed burn reconstruction implant. According to some embodiments described herein, the soft tissue implant is for implantation beneath or within the soft tissue described herein and for augmentation, reconstruction, replacement, and / or regeneration of the soft tissue or a body organ comprising the soft tissue.

[0436] According to some of the embodiments described herein, the soft tissue implants described herein further comprise a matrix, as described in any of the embodiments herein. In some of these embodiments, the matrix fills at least a portion of the interior cavity of the composite structure.

[0437] According to some of these embodiments, the matrix comprises rh collagen as described in any of the embodiments herein, and optionally, in addition to rh collagen, one or more ECM components as described in any of the embodiments herein, optionally an integrin-binding material as described in any of the embodiments herein and any combination thereof, and optionally further comprises biological material (e.g., cells, cellular components, and / or adipose tissue or adipose extract), as described in more detail below.

[0438] According to some of the embodiments described herein, the soft tissue implant is prepared by additive manufacturing of a composite scaffold, e.g., by bioprinting, as described herein in any of the embodiments and any combination thereof.

[0439] The soft tissue implants described herein can be prepared by dispensing at least one curable formulation as described in any of the embodiments herein, and sequentially forming multiple layers in a scaffold configuration pattern to form a composite scaffold as described herein in any of the embodiments and any combination thereof.

[0440] For at least some of these layers, as described herein in any embodiment and any combination thereof, a formulation is dispensed that includes recombinant human collagen having at least one curable group, a synthetic polymer having at least one curable group, and optionally an ECM component having a curable group and / or an integrin-binding material having at least one curable material.

[0441] According to some embodiments, preparing the implant further comprises injecting a matrix as described herein in any of the embodiments and any combination thereof into at least the internal cavity of the scaffold, optionally preferably through an injection port in the scaffold.

[0442] In some embodiments, a method for preparing a 3D bioprinted degradable implant includes, after preparing a bioprinted composite scaffold, maintaining the composite scaffold or a composite scaffold comprising a tissue matrix as described herein in a sterile medium. In some embodiments, maintaining the scaffold in a sterile medium, such as sterile phosphate-buffered saline (PBS) or Dulbecco's modified Eagle's medium (DMEM), can include maintaining the bioprinted 3D scaffold in an environment that is free of greater than 99% of viable microorganisms. One of skill in the art will appreciate that the term "sterile medium" can encompass a medium that is substantially free of viable microorganisms.

[0443] In some embodiments, the method of preparing an implant includes a sterilization step. In some embodiments, the sterilization includes the use of ethylene oxide (EtO) sterilization. In this case, the bioprinted scaffold is dried and exposed to EtO by standard procedures.

[0444] In some embodiments, a method of making an implant comprises bioprinting a composite scaffold and, optionally, filling the interior cavities of the scaffold with a matrix as described herein, wherein the bioprinting and / or filling is performed under sterile conditions.

[0445] An exemplary method for preparing an implant as described herein in any of the embodiments and any combination thereof includes: Bioprinting a composite scaffold according to embodiments described herein (preferably under sterile conditions); Drying the scaffold if necessary (e.g., removing liquid by freeze-drying); Optionally sterilizing the scaffold described herein; Optionally, filling the internal cavity of the scaffold (preferably through one or more injection ports) with a matrix as described herein (preferably under sterile conditions) as described in any of the embodiments and any combination thereof herein.

[0446] matrix: In some embodiments, the scaffold comprises a matrix within at least the interior cavity, the matrix comprising at least one extracellular matrix (ECM) component and cells or adipose tissue, or a combination thereof. In some embodiments, the scaffold comprises a matrix as described herein. In some embodiments, the matrix comprises at least one ECM component. In some embodiments, the ECM component comprises rh collagen, HA, fibronectin, heparin, elastin, or laminin, or any combination thereof. In some embodiments, the matrix comprises cells. In some embodiments, the cells comprise pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells, or adipocytes, or any combination thereof. In some embodiments, the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue. In some embodiments, the matrix comprises adipose tissue, e.g., minimally processed adipose tissue. In some embodiments, the matrix comprises an adipose extract, e.g., a minimally processed adipose extract. In some embodiments, the volume of the matrix is ​​between about 5 mL and about 300 mL.

[0447] According to some of the embodiments described herein, the matrix comprises rh collagen, as described herein in any of the embodiments and any combination thereof. In some of these embodiments, the rh collagen does not have attached thereto a curable group or moiety as described herein. Optionally, the rh collagen is a curable rh collagen as described herein.

[0448] In some of the embodiments described herein, the rh collagen is crosslinked fibrillar collagen as described herein, rh collagen-derived particles as described herein (e.g., in the form of rh collagen and / or rh gelatin nanoparticles), and mixtures of any of these.

[0449] In some of these embodiments, the matrix comprises crosslinked fibrillar collagen as described herein and particulate rh collagen and / or rh gelatin (e.g., in the form of nanoparticles). In some of these embodiments, the weight ratio of the crosslinked fibrillar collagen as described herein to the particulate rh collagen and / or rh gelatin (e.g., in the form of nanoparticles) is about 5:1 to about 5:1, including intermediate values ​​and subranges therebetween. In some of these embodiments, the ratio is 3:1 to 1:3, or 2:1 to 1:2, or 2:1 to 1:1, or 3:2 to 2:1, or 3:2 to 1:1, including intermediate values ​​and subranges therebetween.

[0450] In some of the embodiments described herein, the rh collagen is or comprises a cross-linked fibrillar collagen as described herein.

[0451] According to some of the embodiments described herein, the matrix further comprises one or more ECM components (e.g., hyaluronic acid, fibronectin, heparin, elastin, or laminin, or any combination thereof) as described herein in any of the embodiments and any combination thereof.

[0452] According to some of these embodiments, the ECM component does not have one or more curable groups or moieties attached, as described in any of the embodiments herein. According to some of these embodiments, the ECM component is a crosslinkable ECM component, and the matrix can optionally further comprise a crosslinker, as described in any of the embodiments herein. In some embodiments, the ECM component is a curable ECM component, as described in any of the embodiments and any combination thereof herein.

[0453] According to some of these embodiments, the ECM components are cross-linked, for example, by a cross-linking agent and / or by inherent functional groups in their chemical structure.

[0454] According to some of the embodiments described herein, the matrix further comprises one or more integrin-binding materials (e.g., RGD-containing materials, e.g., RGD4C) as described herein in any of the embodiments and any combination thereof.

[0455] According to some of these embodiments, the RGD-containing material does not have one or more curable groups or moieties attached, as described in any of the embodiments herein. According to some of these embodiments, the RGD-containing material is crosslinkable, e.g., comprises one or more cysteine ​​residues capable of forming disulfide bonds. In some embodiments, the matrix can optionally further comprise a crosslinker, as described in any of the embodiments herein. In some embodiments, the RGD-containing material is a curable RGD-containing material, as described in any of the embodiments and any combination thereof herein.

[0456] In some embodiments, the ECM components include rh collagen, hyaluronic acid (HA), fibronectin, heparin, elastin, or laminin, or any combination thereof. In some embodiments, the ECM components include collagen. In some embodiments, the ECM components include collagen as described herein. In some embodiments, the collagen includes human collagen. In some embodiments, the collagen includes recombinant human collagen (rh collagen). In some embodiments, the collagen includes plant-derived collagen. In some embodiments, the collagen includes plant-derived rh collagen. In some embodiments, the collagen includes cross-linked collagen. In some embodiments, the collagen includes modified collagen. In some embodiments, the rh collagen includes cross-linked plant-derived human collagen. In some embodiments, the rh collagen includes plant-derived human collagen as described herein.

[0457] In some embodiments, the ECM components comprise fragmented ECM components. In some embodiments, the ECM components comprise hyaluronic acid (HA). In some embodiments, the HA comprises modified HA. Addition of methacrylate groups to hyaluronic acid (HA) results in photocurable hyaluronic acid-methacrylate (HAMA or MA-HA). In some embodiments, the HA comprises modified HA or a photopolymerizable modified derivative thereof. In some embodiments, the HA comprises methacrylated HA. In some embodiments, the HA comprises thiolated HA. In some embodiments, the HA comprises crosslinked HA.

[0458] In some embodiments, the ECM components comprise modified fibronectin. In some embodiments, the ECM components comprise modified heparin. In some embodiments, the ECM components comprise modified elastin. In some embodiments, the ECM components comprise modified laminin.

[0459] In some embodiments, the matrix further comprises a crosslinker. In some embodiments, the crosslinker comprises a photocurable crosslinker. In some embodiments, the crosslinker comprises SR9035 (ethoxylated (15) trimethylolpropane triacrylate). In some embodiments, the crosslinker comprises a 4-arm PEG-thiol, an 8-arm PEG-thiol, a 4-arm PEG-acrylate, or an 8-arm PEG-acrylate.

[0460] In some embodiments, the crosslinking agent is not necessarily photocurable, but is capable of crosslinking ECM components or any other components on the matrix when subjected to appropriate reaction conditions.

[0461] In some embodiments, the ECM component comprises fibronectin or a functional fragment thereof. In some embodiments, the ECM component comprises heparin or a functional fragment thereof. In some embodiments, the ECM component comprises elastin or a functional fragment thereof. In some embodiments, the ECM component comprises laminin or a functional fragment thereof. Those skilled in the art will appreciate that laminin surrounds adipocytes and is important in preadipocyte adipogenesis and adipocyte lipogenesis.

[0462] In some embodiments, the matrix comprises two different ECM components, three different ECM components, four different ECM components, or five different ECM components, hi some embodiments, the matrix comprises at least two different ECM components, at least three different ECM components, at least four different ECM components, or at least five different ECM components.

[0463] In some embodiments, the ECM components are decellularized (e.g., as described in U.S. Patent Publication No. 2019 / 0022017, the disclosure of which is incorporated herein by reference). In some embodiments, the ECM components are prepared from desired cells and native decellularized ECM. The native tissue is washed with PBS to remove any residual blood. The tissue is sliced ​​and incubated in a hyperhypotonic solution of NaCl. The slices are then incubated with trypsin-EDTA 0.05% for 24 hours (repeated twice). To remove chemicals, the tissue is then washed several times with Triton X-100 + ammonium hydroxide solution, and finally with phosphate-buffered saline (PBS) for several 48-hour wash cycles until no residual bubbles remain. The ECM is sterilized by washing with ethanol (70%) for 2 hours, followed by two washes with double-distilled water. Lyophilized ECM and protease enzymes (e.g., pepsin and / or collagenase and / or trypsin) of varying concentrations and bioactivities were mixed in 15 mL of 0.05 M to 0.2 M HCl and stirred at room temperature (25 °C) for 48 hours. The resulting viscous solution of digested ECM had a pH of approximately 3.0 to 4.0. Depending on the enzyme, the enzyme activity was irreversibly inactivated; for example, in the case of pepsin, the pH rose to 7.4. The solubilized matrix retains ECM proteins and peptide fragments, thus preserving the biochemical components necessary for cell-matrix interactions. The ECM was considered fully solubilized when no particles were detected in the solution.

[0464] Adipose tissue ECM contains large, complex proteins that exhibit distinct domains, the sequences and arrangements of which are highly conserved. Collagen is a key structural protein in the ECM and is the most abundant protein in the ECM. Adipose tissue ECM regulates and promotes cell differentiation, proliferation, survival, and importantly, physiological function. In some embodiments, the ECM components comprise adipose tissue ECM. In some embodiments, the ECM components comprise allogeneic adipose tissue ECM. In some embodiments, the ECM components comprise autologous adipose tissue ECM. In some embodiments, the adipose tissue ECM comprises types I-VII collagen, laminin, fibronectin, elastin, and glycosaminoglycans (GAGs).

[0465] According to some of the embodiments described herein, the matrix comprises rh collagen, optionally other ECM components and / or integrin-binding materials as described herein, and optionally one or more crosslinkers, and can further comprise cellular components, cells and / or adipose tissue.

[0466] According to some embodiments described herein, the matrix comprises rh collagen and, optionally, other ECM components and / or integrin-binding materials, as described herein, wherein at least one of these components is cross-linked. In some embodiments, at least two of these components are cross-linked to one another. In some embodiments, the ECM components are cross-linked by a cross-linking agent.

[0467] In some embodiments, such matrices can be used to infuse the composite scaffolds described herein.

[0468] According to one aspect of some embodiments of the present invention, there is provided a matrix comprising at least one extracellular matrix (ECM) component and cells or adipose tissue, or a combination of cells and / or adipose tissue. In some embodiments, the matrix comprises at least one extracellular matrix (ECM) component. In some embodiments, the matrix comprises rh collagen, alone or in combination with additional ECM components, and comprises cells and / or adipose tissue.

[0469] According to some of the embodiments described herein, the matrices described herein are injectable matrices.

[0470] In some embodiments, such matrices can be used for injection into a soft tissue site to be treated in a subject in need thereof, as described herein.

[0471] In some embodiments, the matrix comprises at least one extracellular matrix (ECM) component and cells as described herein in any of the embodiments, hi some embodiments, the cells comprise pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells, or adipocytes, or any combination thereof.

[0472] In some embodiments, the matrix comprises pericytes. Those skilled in the art will appreciate that the term "pericytes" can encompass spatially isolated contractile cells that surround endothelial cells that line the body's blood vessels, such as capillaries.

[0473] In some embodiments, the matrix comprises adipose-derived stem cells. In some embodiments, the matrix comprises adipocytes. In some embodiments, the matrix comprises preadipocytes. Those skilled in the art will understand that the term "adipocytes," also known as lipid cells, can encompass cells that contain lipid droplets.

[0474] In some embodiments, the matrix comprises endothelial cells. Those skilled in the art will further appreciate that the term "endothelial cells" can include cells of the endothelium that line the surfaces of body cavities, such as blood vessels, lymphatic vessels, and capillaries.

[0475] In some embodiments, the matrix comprises hematopoietic cells. Those skilled in the art will understand that the term "hematopoietic cells" can encompass blood cells, hematopoietic stem cells that can differentiate into red blood cells, and hematopoietic progenitor cells.

[0476] In some embodiments, the matrix comprises progenitor cells. Those skilled in the art will appreciate that the term "progenitor cells" can encompass unipotent cells that are committed to differentiating into specific types of cells and have limited or no self-renewal capacity.

[0477] In some embodiments, the cells comprise autologous cells. In some embodiments, the cells comprise allogeneic cells. Thus, in some embodiments, the cells can be obtained from a subject and the matrix administered to the same subject. In some other embodiments, the cells can be obtained from a matched donor (e.g., a subject with a matching blood type).

[0478] Autologous or allogeneic cells and cellular components can be obtained using methods well known and recognized in the art for extracting and preparing the cells disclosed herein, for example, using the method shown schematically in FIG.

[0479] In some further embodiments, the matrix comprises adipose tissue. Those skilled in the art will appreciate that the term "adipose tissue" can encompass connective tissue composed of multiple cell types, including adipocytes and microvascular cells. In some embodiments, adipose tissue specifically comprises stem cells and endothelial progenitor cells. Those skilled in the art will appreciate that the term "stem cells" can encompass pluripotent cells that have the potential to differentiate into various cell types and have the ability to self-renew.

[0480] In some embodiments, the adipose tissue comprises an adipose extract. Those skilled in the art will understand that the term "adipose extract" refers to adipose (including the connective tissue that stores fat). In some embodiments, the adipose extract, fat cells and / or tissue can be obtained, for example, by suction defatting, liposuction, and / or direct excision. In some embodiments, the adipose extract or adipose tissue is extracted from various regions of the body, including, for example, the abdomen, thighs, buttocks, arms, and neck.

[0481] In some embodiments, the adipose tissue or adipose extract is homogenized. In some embodiments, the adipose extract comprises a homogenized adipose extract. In some embodiments, the adipose extract comprises a minimally processed adipose extract. In some embodiments, the adipose extract comprises a minimally processed homogenized adipose extract, as described herein and in the Examples section below. In some embodiments, the adipose tissue or adipose extract is mechanically comminuted, e.g., minced. In some embodiments, the adipose tissue or adipose extract is comminuted to about 0.5 mm. 2 ~5mm 2 In some embodiments, the adipose tissue or adipose extract is minced or pulverized to allow for injection into an implant scaffold or soft tissue.

[0482] Those skilled in the art will appreciate that the term "homogenization" can encompass producing particles that are substantially similar in size and composition.

[0483] In some embodiments, the adipose tissue described in any of the embodiments herein comprises autologous adipose tissue. In some embodiments, the adipose tissue described in any of the embodiments herein comprises allogeneic adipose tissue. In some embodiments, the adipose tissue can be obtained from a subject and administered to the same subject. In some other embodiments, the adipose tissue can be obtained from a matched donor (e.g., a subject with a matching blood type). Those skilled in the art will also be familiar with available methods for extracting and preparing adipose tissue, such as the methods disclosed herein.

[0484] In some embodiments, the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue (e.g., autologous adipose tissue). In some embodiments, the matrix comprises SVF isolated from adipose tissue (e.g., autologous adipose tissue). In some embodiments, the SVF comprises pericytes, adipose-derived stem cells, preadipocytes, endothelial cells and progenitor cells, and hematopoietic cells, including monocytes and macrophages. In some embodiments, the SVF is transplanted as part of the matrix. SVF placed within a 3D collagen scaffold has the ability to reorganize into 3D organoids in vitro and develop capillary networks, suggesting that SVF cells promote vascular network formation and angiogenesis. One skilled in the art will appreciate that there are various methods for extracting SVF. In one embodiment, a method for SVF cell extraction is described in Example 1 and is shown schematically in Figure 9. In some embodiments, the SVF can be obtained from an autologous or xenogeneic subject.

[0485] In some embodiments, the adipose tissue comprises a fat extract isolated from adipose tissue (e.g., autologous adipose tissue) and minimally processed (e.g., not subjected to enzymatic treatment). In some of these embodiments, the fat extract is a homogenized fat extract.

[0486] In some embodiments, the matrix comprises at least one extracellular matrix (ECM) component and cells or adipose tissue, or a combination of cells and adipose tissue, as described in any of the embodiments herein. Thus, in some embodiments, the matrix comprises at least one ECM component and cells. In some embodiments, the matrix comprises at least one ECM component and adipose tissue. Furthermore, in some embodiments, the matrix comprises at least one ECM component, cells, and adipose tissue.

[0487] In some embodiments, the matrix comprises rh collagen, HA, fibronectin, heparin, elastin, or laminin, or any combination thereof, and cells. In some embodiments, the matrix comprises rh collagen, HA, fibronectin, heparin, elastin, or laminin, or any combination thereof, and adipose tissue. In some embodiments, the matrix comprises rh collagen, HA, fibronectin, heparin, elastin, or laminin, or any combination thereof, and cells and adipose tissue. In some embodiments, the matrix comprises rh collagen, HA, fibronectin, heparin, elastin, or laminin, or any combination thereof, and SVF. In some embodiments, the matrix comprises rh collagen, HA, fibronectin, heparin, elastin, or laminin, or any combination thereof, and minimally processed fat extract.

[0488] In some embodiments, the ratio of ECM components to cells or adipose tissue in the matrix is ​​about 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5. In some embodiments, the ratio of ECM components to cells or adipose tissue is 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5.

[0489] In some embodiments, the matrix comprises at least one extracellular matrix (ECM) component and cells or adipose tissue, or a combination thereof, wherein the ECM component comprises rh collagen and optionally comprises HA, fibronectin, heparin, elastin and / or laminin, or any combination thereof, and the cells comprise pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells or adipocytes, or any combination thereof, or the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue.

[0490] Those skilled in the art will understand that the terms "injection" or "injectable" encompass, in some embodiments, any in vivo insertion or introduction of a matrix into a subject or scaffold as described herein, and in other embodiments, in vitro insertion or introduction of a matrix into a scaffold. In some embodiments, the matrix is ​​injected into the scaffold prior to implantation into a subject. In some embodiments, the matrix is ​​injected into the scaffold after implantation into a subject. In some embodiments, the matrix is ​​injected directly into a subject. In some embodiments, the use of the matrices described herein is for tissue regeneration. In some embodiments, the use of the matrices described herein is for adipose tissue regeneration.

[0491] How to use the matrix: The matrices described herein can be used in methods of preparing and / or using the soft tissue implants described herein in any of the embodiments and any combination thereof. In some embodiments, the method of preparing a degradable soft tissue implant comprises injecting a matrix into at least the interior cavity of a scaffold as detailed herein.

[0492] In some embodiments, the matrix comprises an injectable matrix. In some embodiments, the matrix is ​​injected into a soft tissue (e.g., breast) implant. In some embodiments, the matrix is ​​injected into the interior cavity of a soft tissue implant scaffold, as described herein.

[0493] In some embodiments, the matrix can be injected into a joint or tissue or surrounding structures and organs of the body. In some embodiments, the tissue comprises soft tissue. In some embodiments, the soft tissue comprises tissue of the subject's face, nose, chin, breast, chin, buttocks, hand, leg, foot, chest, lip, or cheek, or other soft tissue described herein. In one embodiment, the soft tissue comprises breast tissue. In some embodiments, the matrix is ​​crosslinked after injection by transcutaneous illumination of the injection site with a light source.

[0494] In some embodiments, disclosed herein are methods for reconstructing or augmenting soft tissue in a subject in need thereof, the methods comprising injecting a matrix into the soft tissue, the matrix comprising at least one extracellular matrix (ECM) component and cells or adipose tissue, or a combination thereof.

[0495] In some embodiments, the matrices disclosed herein effectively comprise ECM components, e.g., rh collagen, and SVF, cells and / or adipose tissue, e.g., homogenized adipose extract (e.g., minimally processed).

[0496] After injection of the matrices described herein into soft tissue, the ECM components promote the proliferation of adipose-derived cellular components, thereby enabling gradual regeneration of the soft tissue. In some embodiments, the injected matrices promote the propagation or formation of new adipose tissue. Thus, the matrices described herein and their use enable soft tissue repair, augmentation, regeneration, or any combination thereof, in a subject.

[0497] In some embodiments, the use of the matrices disclosed herein promotes the regeneration of new tissue. In some embodiments, the use of the matrices disclosed herein promotes the regeneration of new adipose tissue. In some embodiments, the use of the matrices disclosed herein is for the regeneration of new tissue. In some embodiments, the use of the matrices disclosed herein is for the regeneration of adipose tissue.

[0498] In some embodiments, a method of promoting soft tissue healing in a subject in need thereof is disclosed, the method comprising injecting a matrix into the soft tissue, the matrix comprising at least one extracellular matrix (ECM) component and cells or adipose tissue, or a combination thereof, as described in any of the embodiments herein.

[0499] In some embodiments, there is provided a method of reconstructing and / or augmenting and / or regenerating soft tissue in a subject in need thereof, the method comprising: injecting a matrix into the soft tissue, the matrix comprising: at least one extracellular matrix (ECM) component; cells or adipose tissue, or a combination thereof wherein the at least one ECM component comprises rh collagen, HA, fibronectin, heparin, elastin, and laminin, or any combination thereof, and the cells comprise pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells, or adipocytes, or any combination thereof, or the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue.

[0500] The components of the matrix are described in detail herein. In some embodiments, a method for reconstructing or augmenting soft tissue in a subject in need thereof utilizes any of the embodiments of the matrix described herein. For example, but not limited to, the rh collagen includes plant-derived human collagen, or includes cross-linked rh collagen or modified rh collagen, or the ECM component is a cross-linked ECM component.

[0501] In some embodiments, the method of using the matrix comprises injecting the matrix into a tissue space below the epidermis. In some embodiments, the method of using the matrix comprises injecting the matrix into a tissue space below the dermis (subcutaneous). In some embodiments, the method of using the matrix comprises injecting the matrix into a tissue space within a joint (for example, but not limited to, an injured joint).

[0502] In some embodiments, the matrices described herein can be used in methods for blocking or filling various lumens and voids just below the skin surface. In some embodiments, the matrices described herein can be used in methods for performing tissue augmentation in a subject (e.g., a human patient) in need of tissue augmentation, where the matrix is ​​introduced to the site of interest (e.g., injected into or at the tissue site in need of augmentation) using methods known in the art.

[0503] Those skilled in the art will understand that the terms "reconstruction" or "augmentation" can be used interchangeably with "regeneration" and "repair" and mean to augment, fill, restore, strengthen or replace soft tissue.

[0504] Those skilled in the art will appreciate that "augmentation" can include repairing, preventing, or alleviating defects (particularly defects due to tissue loss or absence) by providing a matrix as described herein to a tissue or augmenting or replacing the tissue with a matrix. Augmentation can also include supplementing a natural structure or feature, i.e., adding constructs to an existing body part to increase the size of, for example, lips, nose, breasts, ears, parts of an organ, chin, cheeks, etc. Thus, tissue augmentation includes filling or reducing grooves, folds, wrinkles, scars, facial dimples, cleft lips, surface wrinkles, etc. in or on the face, neck, hands, feet, fingers, toes, etc.; correction of minor deformities of the hands, feet, fingers, toes, etc. due to aging or disease; augmentation of the vocal cords and glottis to rehabilitate speech; skin filling of sleep and expression wrinkles; replacement of dermis and subcutaneous tissue lost due to aging; lip augmentation; filling of wrinkles around the eyes and orbital sulci; breast augmentation, chin augmentation, cheek and / or nose augmentation; filling of depressions in the skin or subcutaneous soft tissue such as those caused by excessive liposuction or other trauma; filling of acne and traumatic scars and fine lines; and filling of nasolabial, naso-brow and suboral sulci.

[0505] In some embodiments, the matrix can be inserted or introduced into a subject using a suitable injection device, examples of which include, but are not limited to, needles, sharpened plastic-tipped applicators, reservoirs, plungers, release systems, and syringes. In some embodiments, devices suitable for injecting the matrix include, but are not limited to, needles, cannulas, sharpened plastic-tipped applicators, reservoirs, stents, plungers, release systems, and syringes. In some embodiments, the matrix can be administered by local injection (e.g., catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration).

[0506] Those skilled in the art will understand that the term "injectable" can describe a matrix that has a texture or viscosity that allows it to flow through an appropriate delivery device, such as a surgical needle, cannula, other surgical instrument, or other delivery means (e.g., instruments used in endoscopic or percutaneous discectomy). The matrices described herein are injectable through an appropriate applicator, such as a catheter, cannula, needle, syringe, tubular device, or the like, as known in the art.

[0507] In one embodiment, a method of using the matrix of the embodiments described herein is disclosed, in which the matrix is ​​injected into a subepidermal tissue space to induce a cell growth-promoting scaffold, the matrix containing at least one ECM component and cells or adipose tissue, or both, to promote healing or replacement of degraded or damaged collagen-containing tissue.

[0508] In some embodiments, the injection site for the matrix comprises soft tissue, including tissue of the face, nose, chin, breast, chin, buttocks, hand, leg, foot, chest, lip or cheek, or any combination thereof.

[0509] In some embodiments, a method of using the matrix comprises injecting a predetermined amount of the matrix into a site of need in a subject in need thereof, where the amount of injected matrix fills a void. In some embodiments, a method of using the matrix comprises injecting a predetermined amount of the matrix into a site of need in a subject in need thereof, where the amount of injected matrix fills a void in or around damaged tissue.

[0510] In some embodiments, the volume of the injection matrix is ​​about 2 mL to about 500 mL, or about 5 mL to about 500 mL, or about 5 mL to about 300 mL, or about 10 mL to about 500 mL, or about 10 mL to about 300 mL, or about 50 mL to about 500 mL, or about 50 mL to about 300 mL, or about 50 mL to about 250 mL, or about 50 mL to about 150 mL, or about 100 mL to about 150 mL (including intermediate values ​​and subranges therebetween). In some embodiments, the volume of the injection matrix is ​​about 20 mL to 50 mL. In some embodiments, the volume of the injection matrix is ​​about 50 mL to 150 mL. In some embodiments, the volume of the injection matrix is ​​about 150 mL to 300 mL.

[0511] In some embodiments, the volume of the injection matrix is ​​about 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 210 mL, 220 mL, 230 mL, 240 mL, 250 mL, 260 mL, 270 mL, 280 mL, 290 mL, 300 mL, or 350 mL. In some embodiments, the volume of the injection matrix is ​​at least 2 mL, at least 3 mL, at least 4 mL, at least 5 mL, at least 6 mL, at least 7 mL, at least 8 mL, at least 9 mL, at least 10 mL, at least 11 mL, at least 12 mL, at least 13 mL, at least 14 mL, at least 15 mL, at least 16 mL, at least 17 mL, at least 18 mL, at least 19 mL, at least 20 mL, at least 25 mL, at least 30 mL, at least 35 mL, at least 40 mL, at least 50 mL, at least 60 mL, at least 70 mL, at least 80 mL, at least 90 mL, at least 100 mL, at least 110 mL, at least 120 mL, at least 130 mL, at least 140 mL, at least 150 mL, at least 160 mL, at least 170 mL, at least 180 mL, at least 190 mL, at least 200 mL, at least 250 mL, at least 300 mL, at least 350 mL, at least 400 mL, at least 500 mL, at least 600 mL, at least 700 mL, at least 800 mL, at least 900 mL, at least 10 ... 0 mL, at least 80 mL, at least 90 mL, at least 100 mL, at least 110 mL, at least 120 mL, at least 130 mL, at least 140 mL, at least 150 mL, at least 160 mL, at least 170 mL, at least 180 mL, at least 190 mL, at least 200 mL, at least 210 mL, at least 220 mL, at least 230 mL, at least 240 mL, at least 250 mL, at least 260 mL, at least 270 mL, at least 280 mL, at least 290 mL, at least 300 mL, or at least 350 mL.

[0512] In some embodiments, the use of a scaffold-independent matrix is ​​to regenerate smaller volumes of tissue than if the matrix were contained within the scaffold, hi some embodiments, the use of a scaffold-independent matrix is ​​to regenerate smaller volumes of tissue that do not rely on the scaffold to provide the shape or dimension of the regenerated tissue.

[0513] In some embodiments, a method of soft tissue reconstruction, augmentation, or regeneration in a subject in need thereof is disclosed, the method comprising injecting a matrix into the soft tissue, the matrix comprising at least one extracellular matrix (ECM) component and cells or adipose tissue, or a combination thereof, the ECM component comprising rh collagen, HA, fibronectin, heparin, elastin, and laminin, or any combination thereof, and the cells comprising pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells, or adipocytes, or any combination thereof, or the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue.

[0514] According to some of the embodiments described herein, there is provided the use of a matrix as described herein (independent of a scaffold) for reconstructing or augmenting or regenerating or repairing or healing soft tissue in a subject in need thereof, wherein the reconstructing or augmenting or regenerating or repairing or healing of the soft tissue is effected by injecting the matrix as described herein into the soft tissue.

[0515] According to some of the embodiments described herein, there is provided the use of a matrix as described herein (independent of a scaffold) in the manufacture of a medicament for reconstructing or augmenting or regenerating or repairing or healing soft tissue in a subject in need thereof, wherein the reconstructing or augmenting or regenerating or repairing or healing of the soft tissue is effected by injecting the matrix as described herein into the soft tissue.

[0516] In some embodiments, the matrix can be used in a variety of cosmetic, plastic, and reconstructive surgery procedures and can be delivered to many different parts of the body (e.g., including, but not limited to, the face, nose, chin, breasts, chin, buttocks, hands, legs, feet, chest, lips, and cheeks). In some embodiments, the matrix can be used to repair damaged soft tissue due to surgery, disease, or trauma. In some embodiments, the matrix is ​​used to fill soft tissue cavities and augment tissue or organs. In some other embodiments, the matrix is ​​used for cosmetic or aesthetic purposes, for example, in filling wrinkles (fine lines) or repairing aging tissue. In some embodiments, injection of the matrix can be into joints / tissues or surrounding structures and organs of the body.

[0517] In some embodiments, the matrix is ​​crosslinked after injection by transdermal irradiation / illumination of the injection site with a light source. In some embodiments of methods of using the matrix, such as for cosmetic or medical applications, the matrix is ​​photopolymerizable by including one or more components that are curable (e.g., photocurable or photopolymerizable), as described in any of the embodiments herein. In some embodiments, the matrix includes crosslinkable rh collagen. In some embodiments, the matrix includes modified rh collagen. In some embodiments, the matrix includes curable rh collagen, as described in any of the embodiments herein. In some embodiments, the matrix includes modified HA, or photopolymerizable or curable or crosslinkable HA, as described in any of the embodiments herein. In some embodiments, the matrix includes modified fibronectin, or photopolymerizable or curable or crosslinkable fibronectin, as described in any of the embodiments herein. In some embodiments, the matrix includes modified ECM components, or photopolymerizable or curable or crosslinkable ECM components, as described in any of the embodiments herein.

[0518] Typically, the matrix is ​​injected into soft tissue, exposed to curing conditions as described herein, and applied to the skin surface, ie, externally to the body or skin, or to the epidermis adjacent to the injection site.

[0519] In some of these embodiments, the method of performing soft tissue reconstruction or augmentation in a subject in need thereof utilizing the injectable matrices described herein further comprises illuminating the injection site with a light source.

[0520] In some embodiments, the matrix further comprises a photoinitiator, as described herein in any of the embodiments and any combination thereof.

[0521] As used herein, an injectable matrix as described in any of the embodiments relating to an injectable matrix for use without an implant is also referred to herein as an injectable formulation.

[0522] According to other embodiments relating to the use of the matrices described herein, the matrix is ​​injected into an implant, for example, into the internal cavity of a composite scaffold as described herein. In some embodiments, the matrix is ​​injected into the scaffold prior to implantation into a subject. In some embodiments, the matrix is ​​injected into the scaffold after implantation into a subject. In some embodiments, the matrix is ​​injected directly into a subject in conjunction with implantation of an implant as described herein. In some embodiments, the matrix is ​​injected into the scaffold via an injection port. In some embodiments, the diameter of the injection port on the scaffold is sized to allow for injection of the matrices described herein. In some embodiments, the diameter of the injection port is sized to allow for injection of ECM components, cells, or tissues as described herein.

[0523] In some embodiments, the matrix is ​​characterized by a viscosity that allows it to be injected through a medical device, for example, as described in any of the embodiments herein.

[0524] In some embodiments, the matrices described herein have a viscosity in the range of 5 to 20 Pa / sec, or 10 to 20 Pa / sec, or 15 to 20 Pa / sec.

[0525] Methods of using degradable soft tissue implants According to some embodiments of the present invention, there is provided a method of implanting a 3D bioprinted biocompatible and degradable breast implant, comprising a composite scaffold as described herein in any of the embodiments and any combination thereof, into a subject in need thereof, the method comprising: (a) implanting the scaffold into the subject; (b) injecting a matrix into the scaffold, as needed, using at least one injection port in the scaffold; and (c) repeating step (b) as needed, wherein the implanted scaffold gradually degrades over time and the degraded scaffold is replaced by newly formed tissue.

[0526] According to some embodiments of the present invention, there is provided a method of replacing or reconstructing or generating or augmenting or repairing or healing soft tissue (e.g., breast tissue) in a subject in need thereof, comprising: (a) implanting an implant as described in any of the embodiments herein at a treatment site in a subject in need thereof; (b) injecting a matrix into the scaffold as needed using at least one injection port in the scaffold; and (c) repeating step (b) as needed, wherein the implanted scaffold gradually degrades over time and the degraded scaffold is replaced by newly formed tissue. According to one aspect of some embodiments of the present invention, there is provided a bioprinted soft tissue implant as described in any of the embodiments and any combination thereof herein for use in augmenting and / or reconstructing and / or regenerating and / or repairing and / or healing soft tissue in a subject in need thereof, the method comprising: Implanting the scaffold into a body organ or cavity where soft tissue augmentation and / or reconstruction and / or regeneration and / or repair and / or healing is desired, and optionally injecting a matrix as described herein in any of the embodiments into at least the internal cavity of the scaffold, preferably through an injection port of the scaffold; An implant is provided comprising:

[0527] The matrix can be injected before or after implantation.

[0528] According to one aspect of some embodiments of the present invention there is provided a method of augmenting and / or reconstructing and / or regenerating and / or repairing and / or healing soft tissue in a subject in need thereof, comprising: Implanting the scaffold into a body organ or cavity where soft tissue augmentation and / or reconstruction and / or regeneration and / or repair and / or healing is desired, and optionally injecting a matrix as described herein in any of the embodiments into at least the internal cavity of the scaffold, preferably through an injection port of the scaffold; A method is provided that includes:

[0529] The matrix can be injected before or after implantation.

[0530] According to an aspect of some embodiments of the present invention, there is provided a soft tissue implant as described herein in any of the embodiments and any combination thereof for use as a medical device for augmenting and / or reconstructing and / or regenerating and / or repairing and / or healing soft tissue in a subject in need thereof, wherein the medical device comprises implanting a scaffold into a body organ or cavity in which soft tissue augmentation and / or reconstruction and / or regeneration and / or repair and / or healing is desired, and optionally injecting a matrix as described herein in any of the embodiments into at least the internal cavity of the scaffold, preferably through an injection port in the scaffold.

[0531] The matrix can be injected before or after implantation.

[0532] According to some embodiments, matrix injection is performed after implantation and repeated as necessary as described herein.

[0533] According to some embodiments, when a matrix is ​​injected into a scaffold, the amount of matrix is ​​from about 5 mL to about 300 mL, as described in any of the embodiments herein.

[0534] According to some of the embodiments described herein, after implantation, the printed vascular network pathway is anastomosed with at least one of the subject's blood vessels, as described herein.

[0535] Details of soft tissue implants comprising composite scaffolds are described above. Such description is fully incorporated herein. Embodiments of soft tissue (e.g., breast) implants comprising composite scaffolds described herein can be used in methods of implanting soft tissue (e.g., breast) implants and methods of use for replacing or reconstructing soft tissue.

[0536] In some embodiments, the composite scaffolds described herein are bioprinted with a size and shape corresponding to the anatomy to be treated. Such parameters can be obtained, for example, from image data, and the method can further include obtaining image data regarding the size and shape of the scaffold and bioprinting the scaffold according to the image data.

[0537] In some embodiments of the method of using a soft tissue implant, a matrix is ​​loaded into the scaffold and injected through at least one injection port prior to implanting the soft tissue implant comprising the composite scaffold into a subject. In some embodiments of the method of using a soft tissue implant, a matrix is ​​loaded into the scaffold prior to implanting the soft tissue implant comprising the composite scaffold into a subject. In some embodiments of the method of using a soft tissue implant, the scaffold is implanted into a subject without being loaded with a matrix. In some embodiments of the method of using a soft tissue implant, the scaffold is implanted into a subject without a matrix that allows for vascularization of the implant. In some embodiments of the method of using a soft tissue implant, the scaffold is implanted into a subject without being loaded with a matrix, and the matrix is ​​loaded into the scaffold about 1 week to 24 weeks after implantation of the soft tissue implant. In some embodiments of the method of using a soft tissue implant, a matrix is ​​injected into the scaffold using at least one injection port in the scaffold after implanting the scaffold into a subject. The ECM components, cells, and / or adipose tissue contained in the matrix provide a basis for new tissue formation. In some embodiments, upon degradation or bioresorption of the composite scaffold, or a combination thereof, cells present within the matrix can proliferate and spread into the porous regions of the scaffold to form new tissue. In some embodiments, upon degradation or bioresorption of the composite scaffold, or a combination thereof, cells within the tissue present within the matrix can proliferate and spread into the porous regions of the scaffold to form new tissue. In some embodiments, upon degradation or bioresorption of the composite scaffold, or a combination thereof, ECM components within the matrix can provide 3D structural support for new tissue formation (e.g., including but not limited to, cell adhesion).

[0538] In some embodiments of the method of using the soft tissue implant, after implantation, the composite scaffold can degrade over time and be replaced with newly formed tissue. In some embodiments of the method of using the soft tissue implant, after implantation, the composite scaffold can degrade over time and be replaced with matrix and newly formed tissue components. In some embodiments, the soft tissue implant degrades over a predetermined period of time. In some embodiments, according to the method of using the soft tissue implant, the implant gradually degrades over 1 month, 3 months, 6 months, 12 months, 24 months, or 36 months, as described above for soft tissue implants.

[0539] In some embodiments of the method of using a soft tissue implant, the composite scaffold degrades up to 36 months after implantation. In some embodiments of the method of using a soft tissue implant, the composite scaffold degrades up to 24 months after implantation. In some embodiments of the method of using a soft tissue implant, the composite scaffold degrades up to 12 months after implantation. In some embodiments of the method of using a soft tissue implant, the composite scaffold degrades between 12 and 36 months after implantation. In some embodiments of the method of using a soft tissue implant, the composite scaffold degrades between 12 and 24 months after implantation. In some embodiments of the method of using a soft tissue implant, the composite scaffold degrades between 6 and 18 months after implantation. In some embodiments of the method of using a soft tissue implant, the composite scaffold degrades between 1 month and 36 months after implantation. In some embodiments of the method of using a soft tissue implant, the composite scaffold degrades between 3 and 24 months after implantation.

[0540] In some embodiments of the method of using the soft tissue implant, the 3D bioprinted biocompatible and degradable soft tissue implant composite scaffold is capable of being implanted in a subject within about 6 months of implantation, within about 7 months of implantation, within about 8 months of implantation, within about 9 months of implantation, within about 10 months of implantation, within about 11 months of implantation, within about 12 months of implantation, within about 13 months of implantation, within about 14 months of implantation, within about 15 months of implantation, within about 16 months of implantation, within about 17 months of implantation, within about 18 months of implantation, or within about 19 months of implantation. , within about 20 months from implantation into the subject, within about 21 months from implantation into the subject, within about 22 months from implantation into the subject, within about 23 months from implantation into the subject, within about 24 months from implantation into the subject, within about 25 months from implantation into the subject, within about 26 months from implantation into the subject, within about 27 months from implantation into the subject, within about 28 months from implantation into the subject, within about 29 months from implantation into the subject, within about 30 months from implantation into the subject, within about 31 months from implantation into the subject, within about 32 months from implantation into the subject, within about 33 months from implantation into the subject, within about 34 months from implantation into the subject, within about 35 months from implantation into the subject, or within about 36 months from implantation into the subject.

[0541] In some embodiments of the method of using the soft tissue implant, the step of injecting the matrix into the scaffold is repeated. In some embodiments of the method of using the soft tissue implant, the matrix is ​​repeatedly injected into the scaffold after implantation into the subject. In some embodiments of the method of using the soft tissue implant, the matrix is ​​injected into the scaffold at least once. In some embodiments of the method of using the soft tissue implant, the matrix is ​​injected into the scaffold 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0542] In some embodiments of the method of using the soft tissue implant, the step of injecting the matrix into the scaffold occurs between 1 week and 24 weeks after implantation into the subject.

[0543] In some embodiments of the method of using a soft tissue implant, the step of injecting the matrix into the scaffold occurs within a maximum of 24 weeks of implantation. In some embodiments of the method of using a soft tissue implant, the step of injecting the matrix into the scaffold occurs within a maximum of 12 weeks of implantation. In some embodiments of the method of using a soft tissue implant, the step of injecting the matrix into the scaffold occurs within a maximum of 12 weeks of implantation. In some embodiments of the method of using a soft tissue implant, the step of injecting the matrix into the scaffold occurs within 1 week to 24 weeks of implantation. In some embodiments of the method of using a soft tissue implant, the step of injecting the matrix into the scaffold occurs within 12 weeks to 24 weeks of implantation. In some embodiments of the method of using a soft tissue implant, the step of injecting the matrix into the scaffold occurs within 6 weeks to 18 weeks of implantation. In some embodiments of the method of using a soft tissue implant, the step of injecting the matrix into the scaffold occurs within 1 week to 12 weeks of implantation. In some embodiments of the method of using the soft tissue implant, the step of injecting the matrix into the scaffold occurs within 3 to 24 weeks of implantation.

[0544] In some embodiments of the method of using the soft tissue implant, the step of injecting the matrix into the scaffold comprises injecting the matrix into the scaffold within about 1 week of implantation into the subject, within about 2 weeks of implantation into the subject, within about 3 weeks of implantation into the subject, within about 4 weeks of implantation into the subject, within about 5 weeks of implantation into the subject, within about 6 weeks of implantation into the subject, within about 7 weeks of implantation into the subject, within about 8 weeks of implantation into the subject, within about 9 weeks of implantation into the subject, within about 10 weeks of implantation into the subject, within about 11 weeks of implantation into the subject, The transplantation is performed within about 12 weeks from transplantation into the subject, within about 13 weeks from transplantation into the subject, within about 14 weeks from transplantation into the subject, within about 15 weeks from transplantation into the subject, within about 16 weeks from transplantation into the subject, within about 17 weeks from transplantation into the subject, within about 18 weeks from transplantation into the subject, within about 19 weeks from transplantation into the subject, within about 20 weeks from transplantation into the subject, within about 21 weeks from transplantation into the subject, within about 22 weeks from transplantation into the subject, within about 23 weeks from transplantation into the subject, or within about 24 weeks from transplantation into the subject.

[0545] In some embodiments of the method for implanting the soft tissue implant, the matrix can be administered by local injection, e.g., catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. In some embodiments of the method for using the soft tissue implant, devices suitable for injecting the matrix include needles, cannulas, sharp plastic tip applicators, reservoirs, stents, plungers, release systems, and syringes.

[0546] In some embodiments, the method of implanting a 3D bioprinted degradable soft tissue implant includes a step of preloading the scaffold prior to implantation step (a), in which the scaffold is preloaded with a matrix comprising at least one ECM component and cells or adipose tissue, or a combination thereof.

[0547] In some embodiments of the method for implanting a soft tissue implant, at least the internal cavity of the scaffold is pre-filled with a matrix. In some embodiments of the method for implanting a soft tissue implant, the matrix comprises at least one extracellular matrix (ECM) component and cells or adipose tissue, or a combination thereof. In some embodiments of the method for implanting a soft tissue implant, the scaffold comprises a matrix as detailed herein. In some embodiments of the method for implanting a soft tissue implant, the matrix comprises at least one ECM component. In some embodiments of the method for implanting a soft tissue implant, the ECM component comprises rh collagen, HA, fibronectin, heparin, elastin, or laminin, or any combination thereof. In some embodiments, the HA comprises modified HA or a photopolymerizable modified derivative thereof. In some embodiments, the HA comprises methacrylated or thiolated HA, as detailed herein.

[0548] In some embodiments of the method of using a soft tissue implant, the matrix comprises cells. In some embodiments of the method of implanting a soft tissue implant, the cells comprise pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells, or adipocytes, or any combination thereof. In some embodiments of the method of implanting a soft tissue implant, the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue. In some embodiments of the method of implanting a soft tissue implant, the matrix comprises adipose tissue. In some embodiments of the method of implanting a soft tissue implant, the adipose tissue comprises a homogenized adipose extract.

[0549] In some embodiments of the method of using a soft tissue implant comprising a composite scaffold, the amount of pre-loaded matrix is ​​as described herein in any of the embodiments.

[0550] Details of the composite scaffolds and matrices are provided above, and these details and embodiments thereof are fully incorporated herein, and the methods of use of the soft tissue implants include embodiments of the soft tissue implants and matrices described herein.

[0551] In some embodiments of the methods for implanting a soft tissue implant described herein, the method includes anastomosing at least one of the printed vascular network pathways with at least one of the subject's blood vessels. Those skilled in the art will understand that the term "anastomosis" can encompass surgically joining a subject's blood vessel to a soft tissue implant or scaffold described herein. In some embodiments, the subject's blood vessel is coupled to the scaffold via at least one of the printed vascular network pathways. In some embodiments, the subject's blood vessel is coupled via the printed vascular network pathway so that cells and tissues within the interior cavity of the scaffold receive a constant supply of oxygen and nutrients. In some embodiments, the vascular network pathway is designed to allow anastomosis to the subject's blood vessel via a surgical procedure.

[0552] In some embodiments of the methods of implanting a soft tissue implant described herein, the method includes anastomosing the printed vascular network pathway with at least one of the subject's blood vessels.

[0553] In some embodiments of the methods for implanting a soft tissue implant described herein, the soft tissue implant comprises a composite scaffold comprising rh collagen and a biocompatible synthetic polymer for injection into a subject in need thereof, the method comprising: (a) implanting the scaffold into the subject, wherein the scaffold comprises a porous lattice, an internal cavity within the scaffold, and at least one injection port connecting the internal cavity to the outermost surface of the scaffold, the injection port being sized to allow insertion of a cannula for cell or tissue injection; (b) optionally, injecting a matrix into the scaffold using the at least one injection port of the scaffold, the matrix comprising at least one extracellular matrix (ECM) component, and cells or adipose tissue, or a combination thereof; and (c) repeating step (b) as needed, wherein the implanted scaffold gradually degrades over time and the degraded scaffold is replaced with newly formed tissue.

[0554] In some embodiments of the methods of implanting a soft tissue implant described herein, the method includes a step of preloading the scaffold prior to implanting step (a), wherein the scaffold is preloaded with a matrix comprising at least one ECM component and cells or adipose tissue, or a combination thereof, wherein the ECM component comprises rh collagen, HA, fibronectin, heparin, elastin, and laminin, or any combination thereof, and the cells comprise pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells, or adipocytes, or any combination thereof, or the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue. In some embodiments of the methods of implanting a soft tissue implant, the adipose tissue comprises a homogenized adipose extract.

[0555] In some embodiments of the methods for implanting a soft tissue implant described herein, implantation promotes vascularization of the implant. In some embodiments of the methods for implanting a soft tissue implant described herein, the scaffold comprises implanted cells and tissue. In some embodiments of the methods for implanting a soft tissue implant described herein, the scaffold is biodegradable, such that the implanted scaffold gradually degrades over time and is eventually replaced by newly formed tissue. In some embodiments of the methods for implanting a soft tissue implant described herein, the scaffold comprises ECM components that promote cell proliferation, cell differentiation, and tissue growth. The ability to properly induce vascularization is important for the success of the implantation procedure. Delays in the delivery of oxygen and nutrients and the removal of waste products can be damaging to the implanted cells and tissue.

[0556] Usage: Any of the curable formulations, composite scaffolds, matrices and implants described herein are for use in augmenting, reconstructing, repairing and / or regenerating soft tissue in a subject in need thereof, by any of the methods described herein.

[0557] When the matrices described herein are used by themselves (without an implant) in accordance with the present embodiments, the matrices are also considered soft tissue fillers for use in applications requiring filling and / or augmenting and / or repairing soft tissue, for example, as described herein.

[0558] "Tissue reconstruction" encompasses the repair or replacement of part or all of a tissue in the body, or the repair or replacement of part or all of an organ. Tissue reconstruction implants or matrices are designed to aid in the tissue reconstruction process. For example, they can take over the role of supporting connective tissue within an organ or body part. In the case of implants that include a three-dimensional scaffold made of biodegradable materials, the implant can temporarily take over the role of supporting connective tissue.

[0559] In some embodiments, the implants described herein are for generating pre-vascularized connective tissue as a recipient site for cell / tissue transplantation, preferably free fat graft transplantation.

[0560] In some embodiments, the methods and uses described herein can be used to treat scarring and deformities following trauma or surgical procedures.

[0561] In some embodiments, the methods and uses described herein can be used to treat soft tissue depressions.

[0562] In some embodiments, the methods and uses described herein can be used to treat congenital deformities.

[0563] In some embodiments, the methods and uses described herein can be used to treat Poland syndrome.

[0564] In some embodiments, the methods and uses described herein can be used to treat Romberg's syndrome.

[0565] In some embodiments, the methods and uses described herein can be used to treat pectus excavatum.

[0566] In some embodiments, the methods and uses described herein can be used to treat structural asymmetries, such as breast asymmetry or hip asymmetry or facial asymmetry.

[0567] In some embodiments, the methods and uses described herein can be used for post-mastectomy and post-lumpectomy breast reconstruction.

[0568] In some embodiments, the methods and uses described herein can be used to treat lipodystrophy.

[0569] In some embodiments, the methods and uses described herein can be used for tissue augmentation (breast, buttocks).

[0570] In some embodiments, the methods and uses described herein can be used to treat any condition requiring cavity filling or improved body contouring.

[0571] kit: According to one aspect of some embodiments of the present invention, one or more curable formulations for forming a composite scaffold according to any of the embodiments and any combination thereof; and a matrix formulation for forming a matrix to be injected into the internal cavity of the scaffold as described in any of the embodiments and any combination thereof.

[0572] In some embodiments, the formulations are individually packaged within the kit, hi some embodiments, the packaging is light-tight and / or air-tight.

[0573] In some embodiments, the kit is specified for the preparation of a soft tissue implant as described in any of the embodiments herein and can include instructions on how to use the formulation in accordance with the embodiments described herein.

[0574] According to one aspect of some embodiments of the present invention, a composite scaffold according to any one of the embodiments and any combination thereof; a matrix formulation for forming a matrix to be injected into the internal cavity of the scaffold, as described in any of the embodiments and any combination thereof; A kit is provided comprising:

[0575] In some embodiments, the scaffold and formulation are packaged separately within the kit, hi some embodiments, the packaging is light- and / or air-impermeable.

[0576] In some embodiments, the kit is specified for use in preparing a soft tissue implant as described in any of the embodiments herein and can include instructions for combining the formulation with a scaffold according to any of the embodiments described herein.

[0577] According to an aspect of some embodiments of the present invention, there is provided a kit comprising an injectable matrix as described in any of the embodiments and any combination thereof.

[0578] In some embodiments, the kit is specified for the preparation of a soft tissue implant as described in any of the embodiments herein and can include instructions on how to use the formulation in accordance with the embodiments described herein.

[0579] In some embodiments, the kit is specified for use as a soft tissue filler, as described in any of the embodiments herein.

[0580] Throughout this specification, when referring to an ECM component (e.g., a hardening ECM component, collagen, rh collagen), according to some embodiments, as described in any of the embodiments and any combination thereof, the ECM component also includes fragments and degradation products thereof.

[0581] As used herein, the term "about" means ±10% or ±5%.

[0582] The terms "comprises," "comprising," "includes," "including," "having," and their conjugations mean "including but not limited to."

[0583] The term "consisting of" means "including and limited to."

[0584] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0585] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" includes a plurality of compounds, and may include mixtures thereof.

[0586] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and is not an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all the possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the magnitude of the range.

[0587] When a range of numerical values ​​is given herein, it is intended to include any recited number (fractional or integer) within the range given. The phrases "ranging between" a first designated number and a second designated number and "ranging from" a first designated number to a second designated number are used interchangeably herein and are intended to include the first designated number and the second designated number, and all fractional and integer numbers therebetween.

[0588] As used herein, the term "method" means manner, means, techniques, and procedures for accomplishing a given task, including, but not limited to, those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine, or those that can be readily developed by practitioners from known manners, means, techniques, and procedures.

[0589] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating the clinical or cosmetic symptoms of a condition, or substantially preventing the appearance of clinical or cosmetic symptoms of a condition.

[0590] As used herein, the term "subject" includes animals, preferably mammals, and more preferably humans. The terms "subject," "individual," or "patient" are used interchangeably herein. In one embodiment, in any of the methods and uses described herein, the subject includes a human subject. In some embodiments, the subject is male. In some embodiments, the subject is female.

[0591] It will be understood that features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in any combination of those features in a single embodiment. Conversely, features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, as appropriate, in other described embodiments of the invention. Features described in the context of various embodiments should not be construed as essential features of that embodiment unless the embodiment is inoperative without that element.

[0592] 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.

[0593] Example Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting manner. [Example]

[0594] Preparation of 3D bioprinted degradable breast implants Implant design and preparation: We designed and fabricated exemplary breast implants containing scaffolds that are vascularizable and gradually degrade over a period of 3-36 months. Generally, the scaffolds were formed by 3D printing using a curable formulation containing a curable rh-collagen-based material, and then optionally filled with a matrix containing extracellular matrix components and / or autologous adipocytes (e.g., adipose extract or SVF in a rh-collagen-based matrix) (schematically shown in Figures 7 and 10).

[0595] Scaffold preparation: 3D breast degradable implant scaffolds were bioprinted using a LabFab desktop printer (3D Systems) using the curable formulations described herein, including methacrylated rh collagen, as described in any of the embodiments herein (Figure 7, top left).

[0596] An exemplary formulation contained methacrylated rh collagen (0.5%), poly(ethylene glycol) diacrylate 700 (PEG-DA, average Mn=700; 0.5-2%), ethoxylated (15) trimethylolpropane triacrylate (commercially available as SR9035; 1.0-2.5%), and N-(2-hydroxyethyl)acrylamide (HEAA; 12.0-40.0%), as well as, optionally, ECM components and / or RGD-containing materials described herein, all in an aqueous carrier (e.g., water).

[0597] Further exemplary formulations included methacrylated rh collagen (0.1-1%), a synthetic polymer having a curable group as described in any of the embodiments herein (5-30%), an ECM component having a curable group as described in any of the embodiments herein, e.g., (meth)acrylated or thiolated fibronectin and / or (meth)acrylated or thiolated heparin (0.001-0.1%), and an integrin-binding material having a curable group (e.g., a cysteine-containing RGD material and / or an RGD-containing material conjugated to PEG-DA as described herein) (in approximately stoichiometric amounts relative to the rh collagen).

[0598] The exemplary 3D-printed scaffold was generally dome-shaped with dimensions of 10 mm x 10 mm x 6 mm and contained pores characterized by dimensions of approximately 0.5 mm x 0.5 mm. Figures 8A-8C show a cross-section (Figure 8A), a top view (Figure 8B), and a side view (Figure 8C) of the printed 3D scaffold, with pore dimensions of 500 microns (micrometers). For example, as shown in Figure 10 and described herein, the scaffold was designed to feature at least one (e.g., two) injection ports and a vascular network in its interior space, and printed vascular network pathways around the edges of the scaffold.

[0599] Further designs of exemplary generally dome-shaped 3D printed scaffolds described herein are shown in FIGS. 14A-1B and 15.

[0600] Material / Matrix Filling: Injectable matrices according to some embodiments of the present invention typically comprise one or more ECM components (e.g., collagen, preferably rh collagen as described herein, hyaluronic acid, fibronectin, heparin, elastin, or laminin, and / or any combination thereof), and optionally further comprise an adipose extract (e.g., autologous adipose extract) as described herein in association with cells and / or adipose tissue. Autologous adipose extract, such as autologous stromal vascular fraction (SVF) (used in in vitro studies) or autologous homogenized adipose extract (used in in vivo studies), is optionally prepared and mixed with the matrix. In some embodiments, the autologous homogenized adipose extract is minimally processed, as described in further detail below.

[0601] Preparation of autologous fat extract: Autologous stromal vascular fractions are typically prepared as shown in Figure 9. Autologous adipose tissue was transferred to a 50 mL conical tube containing ice-cold PBS. The capped conical tube was vigorously shaken to wash the fat, then transferred to a new tube. This washing process was repeated until the solution was clear. The adipose tissue was then finely minced and transferred to a 50 mL centrifuge tube. 0.1% collagenase solution (1 mg / mL) was added to the sample, and the tissue was incubated at 37°C for 60 minutes with gentle shaking (60 cycles / min). Complete medium (CM) (MEM containing 10% fetal bovine serum and P / S) was added to the tube to neutralize the digestion, followed by inversion to mix. After collecting the material by centrifugation, three distinct layers were clearly visible in the tube. The top layer contained oil and fat. The middle layer was aqueous and appeared red / transparent. The stromal vesicle fraction (SVF) was present as a brownish pellet at the bottom of the tube, separate from the other layers. The SVF was resuspended in PBS to remove collagenase, centrifuged at 700 × g for 5 minutes at room temperature, resuspended in PBS, and collected by centrifugation. The SVF pellet was resuspended in 5 mL of PBS, filtered to remove large tissue particles, pipetted onto a 100 μm mesh filter placed over a new 50 mL centrifuge tube, and filtered by gravity flow. The filter was rinsed with 5 mL of PBS and centrifuged at 280 × g for 5 minutes at room temperature. The supernatant was removed, and the pellet was resuspended in an appropriate volume of CM. The resulting solution essentially consisted of a single-cell suspension of SVF cells. Viable cells were counted using trypan blue exclusion.

[0602] Self-homogenized fat extracts were prepared in a manner similar to that shown in Figure 9 and the accompanying description, except that the isolation procedure was stopped with mechanical mincing / homogenization of the fat pads without enzymatic digestion, thereby obtaining a minimally processed extract.

[0603] Matrix filling: The adipose extract was mixed with cross-linked fibrillar rh collagen (e.g., EDC X-conjugated fibrillar rh collagen, such as X-Fb-rhCol-EDC20, described in Example 4 below) to yield a total of approximately 500 microliters of adipose extract and rh collagen-containing matrix for injection into the scaffolds, as shown in Figure 10. Scaffolds were loaded using a 1 mL syringe equipped with an 18G needle.

[0604] In the exemplary matrix, the weight ratio of rh collagen to fat extract was 1:3.

[0605] Using this method, biocompatible and degradable scaffolds were fabricated by 3D printing.

[0606] Characterization: The mechanical properties of implants prepared according to some embodiments of the present invention are determined using methods known and accepted in the art, as illustrated in Figures 1A-6 of the Background Art (adapted from Brandon et al. (2019) Bioengineering (Basel). 6(2):43).

[0607] 1A-1B of the Background Art show an example of a compression test between two parallel plates used to characterize the mechanical properties of implants, FIG. 2 of the Background Art shows an exemplary graph of analytical test load as a function of projected strain, FIG. 3 of the Background Art shows an exemplary graph of analytical test load as a function of diametral strain, FIG. 4 of the Background Art shows an exemplary graph of analytical test load as a function of areal strain, FIG. 5 of the Background Art shows a setup for analyzing local strain at very low compressive loads ("pinch" test), and FIG. 6 of the Background Art shows an exemplary graph of results obtained in the "pinch" test.

[0608] Further details are provided in Example 3 below. [Example]

[0609] Animal model studies An example of a typical animal study is shown in Figure 11. 3D bioprinted degradable breast implants are implanted into a rat model under various conditions (e.g., with or without pre-loading or post-injection of the scaffold with matrix (SVF or adipose extract in a rh collagen-based matrix)). The implanted breast implants are then evaluated for safety and status at various time points.

[0610] More specifically, the 3D bioprinted degradable scaffold is printed using recombinant human collagen (rh collagen) in combination with the biocompatible synthetic polymers described herein. In one embodiment, the scaffold is generally dome-shaped and includes an injection port, an internal cavity, and a printed vascular network pathway. The 3D bioprinted degradable scaffold is sterilized with EtO prior to implantation.

[0611] i. 3D bioprinted degradable scaffolds are implanted into the backs of rats. Two to four different implant configurations are evaluated, and each rat is implanted with one scaffold engineered to match one of these configurations. The four possible configurations are: empty 3D bioprinted scaffold (i.e., no internal matrix or homogenous adipocytes injected); ii. 3D bioprinted scaffolds injected with matrix but not homogenous adipocytes. iii. 3D bioprinted scaffolds infused with homogeneous adipocyte-loaded matrix. iv. Injection of matrix and homogenous adipocytes without using a 3D bioprinted scaffold.

[0612] The injection of the matrix with or without homogeneous adipocytes occurs before or after implantation (see Figure 11).

[0613] The effect of implantation on the general health of the rats is assessed, as well as the degradation of the scaffold over time and tissue regeneration and cell distribution throughout the scaffold, using, for example, histological measurements and / or imaging.

[0614] Preliminary studies: A pilot animal study was conducted in a subcutaneous rat model. 3D-printed rhcollagen-based scaffolds were loaded (by injection) with a filler containing cross-linked fibrous rhcollagen-EDC20 with or without adipose extract, and the loaded scaffolds were implanted into subcutaneous pockets created on the backs of rats, as shown in Figures 12A-12C.

[0615] The rat subcutaneous model is a common model for estimating the biocompatibility and regenerative potential of implants combined with artificial scaffolds for soft tissue augmentation.

[0616] Research purpose: The primary objective of this study was to evaluate the ability of the implant and soft tissue filler matrix to maintain the viability of the adipose extract and induce new adipose tissue regeneration at the implantation site in a subcutaneous rat model. The study included histological evaluation to assess tissue integration and regeneration over time, and the formation of a well-functioning neovascular network.

[0617] The primary endpoints were signs of tissue regeneration and integration, and vascularization at the implant / injectable soft filler site.

[0618] The secondary endpoint was the volume retention of the injectable soft filler over time.

[0619] Test articles and materials: Scaffolds were prepared using formulations containing methacrylated rh collagen (0.5%), PEGDA (0.5-2%), SR9035 (1.0-2.5%), and HEAA (12.0-40.0%) as described herein (see Example 1, Figures 7 and 10). The resulting 3D-printed implants were sterilized with ETO.

[0620] 20 mM EDC X-conjugated fibrillar rhcollagen (X-Fb-rhCol-EDC20) was used as an injectable filler.

[0621] The filler was divided into syringes, lyophilized, and sterilized with ETO.

[0622] The fat extract for injection was isolated and prepared on the day of the experiment as described above in Example 1. The composition of the test article is detailed in Table 1.

[0623] [Table 1]

[0624] Study design: The experimental model was subcutaneous implantation into the backs of Sprague-Dawley rats. Groups A and B each received 3D-printed implants in two opposing subcutaneous pockets.

[0625] A total of 6 animals were used in all experimental groups. Animals were sacrificed 4 weeks after implantation / infusion.

[0626] Two additional rats were sacrificed to isolate fat grafts for implants in Group B.

[0627] Histological evaluation: At sacrifice, the implantation sites were exposed and evaluated macroscopically. The implants were excised, fixed in 4% PFA, and subjected to histopathological evaluation.

[0628] Animal test systems: Species / strain: Hsd: Sprague Dawley® SD® Supplier: Harlan Laboratories Israel, Ltd. Gender: Female Total number of animals: 6 for research + 2 for fat collection Age: 10 weeks old Body weight: Approximately 230 grams at the start of the study. Animal weight fluctuations at the start of treatment did not exceed ±20% of the mean body weight. Animal health: The health of animals used in this study will be inspected upon arrival. Only healthy animals will be acclimatized to laboratory conditions and used in the study. Acclimatization: 7 days Housing: Animals were housed in IVC cages in a dedicated HVAC (heat, ventilation, and air-conditioned) animal facility at a temperature of 22 ± 2°C and 55 ± 15% RH. Temperature and humidity were continuously monitored. Food and water: Animals were fed a commercial rodent diet (Harlan Teklad TRM Ra / Mouse Diet) ad libitum and had free access to autoclaved water, supplied to each cage via polysulfone bottles equipped with stainless steel sipper tubes. Environment: Facility is not exposed to external light and is maintained on an automated alternating cycle of 12 hours light and 12 hours dark. Identification: Each cage is labeled with details relating to the study name, animal number, and treatment group. Termination: Animals were euthanized by CO2 asphyxiation at the end of the study.

[0629] Study Procedures Fat graft preparation: On the day of the experiment, adipose tissue was harvested from five rats by SIA. The fat was kept sterile and transported on ice. Adipose extract for injection was prepared in a biological safety hood. The adipose tissue was divided into two 5 mL syringes and homogenized using an additional syringe. After homogenization, the syringes were centrifuged at 1000 g for 1 minute. Phase separation was clearly observed. The upper oil fraction was easily removed. The remaining adipose extract was mixed, divided into multiple syringes (3 mL / syringe), and mixed with rh collagen bulking agent (3:1).

[0630] Analgesia and Anesthesia: Animals were sedated using 2% isoflurane mixed with 3% O2, shaved, and disinfected with 70% ethanol.

[0631] Porting Procedure: Animals in groups A and B were sedated and shaved, and a single midline incision was made in the rat's spine. Subcutaneous pockets were created in both flanks, and one scaffold was placed in each pocket. Prior to implantation, the scaffolds were injected with either an rhCol-based matrix containing adipose extract (group B) or an rhCol-based matrix without adipose extract (group A).

[0632] The implant design and dimensions are shown in Figures 8A-8D and described herein. Approximately 500 microliters of matrix was injected into the dome through one of the designated orifices (see Figure 10) using an 18G needle, as shown in Figure 12A. After implantation (Figure 12B), the wound was closed with a clamp, as shown in Figure 12C.

[0633] Each animal was treated with a maximum of 5 mg of rh collagen.

[0634] Post-operative care: All animals were checked twice daily for morbidity and mortality throughout the study period.

[0635] Organ / Tissue Collection and Fixation: At sacrifice, the graft / injection sites were exposed and evaluated macroscopically. The blebs and implants were excised along with the overlying skin and fixed in 4% PFA.

[0636] Histological measurements: The harvested and fixed samples were subjected to staining and histological evaluation.

[0637] Paraffin blocks were sectioned at approximately 4 microns in thickness, mounted on glass slides, and stained with hematoxylin and eosin (H&E) for general histological evaluation and Masson's trichrome (MT) for evaluation of fibrosis.

[0638] Photographs were taken using a microscope (Olympus BX60, serial number 7D04032) with an objective magnification of ×1.25 and ×10, using a microscope camera (Olympus DP73, serial number OH05504). Photographs were taken only for pathological changes and representative animals.

[0639] All slides were reviewed by a single pathologist.

[0640] A scoring system for histological evaluation (from ISO 10993 part 6) was selected for semi-qualitative evaluation.

[0641] result: The implants in both groups remained intact 4 weeks after implantation. Highly vascularized implants were visible, with extensive vasculature observed in the immediate vicinity of the implants.

[0642] The main parameters extracted from the histological measurements are summarized in Figure 13 A. The inflammation score was calculated as the sum of the scores of all inflammatory cells evaluated (polymorphonuclear cells (PMCs), lymphocytes, plasma cells, macrophages, and giant cells).

[0643] Representative histological images of both implant types are shown in Figures 13B-13E.

[0644] As shown in Figures 13A-13E, high tissue ingrowth was achieved within the scaffold and between the scaffold strands in both groups. Tissue integration was low in both groups, which is likely due to encapsulation and fibrosis. Necrosis was observed in only one implantation site in Group B, which is likely due to sample contamination. Fatty infiltration was mild and inconsequential. Regarding inflammation, the 3D scaffold combined with rh collagen alone (Group A) had a low rate of inflammation, as evidenced by the low presence of mononuclear inflammatory cells.

[0645] Overall, this study found that 3D-printed scaffolds showed promising results in terms of tissue ingrowth and regeneration without any significant adverse tissue reactions in all study groups.

[0646] Specifically, the 3D-printed scaffolds remained intact 4 weeks after implantation, and good tissue ingrowth was achieved within the inner compartment and within the printed strands in both groups. [Example]

[0647] Implant characterization and design The breast implants described herein are designed to degrade over time, with the interior cavity of the implant being replaced by newly formed tissue, although there is an extended timeframe during which the implant remains present within the subject prior to and during the degradation period. The mechanical properties of the implant provide the strength and durability of the degradable implants described herein and play an important role in promoting tissue regeneration to replace the degradable implant over time.

[0648] Ideally, the methods used to analyze the mechanical properties of the scaffold / implant should simulate in vivo conditions, or as close as possible to in vivo conditions, for the target tissue (the tissue that will be augmented by the scaffold implant). Likewise, the scaffold should possess mechanical properties that efficiently promote tissue growth.

[0649] In some embodiments, mechanical properties analyzed include, but are not limited to, multidimensional strain and tangent modulus, shape stability, implant mobility, and fatigue failure properties. This information can provide a basis for indicating the durability of an implant when compressed in vivo. Information indicating dimensional strain can be useful because it represents the geometric or shape changes that may occur in response to compressive loading. Tangent modulus is useful for describing the behavior of a material stressed beyond its elastic region. Tangent modulus quantifies the changes that occur when a material begins to yield to stress or strain by "softening" or "hardening." For example, one skilled in the art can use tangent modulus to quantify the buckling failure of a breast implant under expected "normal" stress conditions.

[0650] In some embodiments, the mechanical properties of the scaffolds can be analyzed using methods currently known in the art. Examples of evaluation methods include those described in Brandon et al., (2019) "New Evaluation Procedure for Multi-Dimensional Mechanical Strains and Tangent Moduli of Breast Implants: IDEAL IMPLANT" and others. RThe methods used include those described in “Structured Breast Implant Compared to Silicone Gel Implants,” Bioengineering, 6:43. Brandon et al. (ibid.) describe a series of mechanical analyses to characterize the mechanical properties of the tested implants, including compression tests between two plates (Figures 1A and 1B) and localized strain (“pinching”) at very low compressive loads (Figure 5). The mechanical analyses described in Brandon et al., 2019 (supra) were performed under both static and dynamic conditions, and, as described there, were performed in the presence and absence of lubricants to test for differences in the presence and absence of frictional forces.

[0651] Multidimensional strain analysis of an implant can be performed, for example, by the method shown in Figures 1A to 1B (but is not limited to this), which can provide results for determining projective distortion, diameter distortion, and area distortion.

[0652] Strain is the rate of change in the dimensions of the implant in response to a load (in this case a compressive load). Lower strain values ​​indicate a more dimensionally stable implant. Using Figures 1A and 1B as a guide, projective, diametric, and areal strains can be calculated using the following formulas:

[0653]

number

[0654] Figures 2-4 show the results of compression tests described in Brandon et al., 2019 (cited above) and relate to "high-cycle, low-cycle, and accidental loading that breast implants may experience over their lifetime" (Brandon et al., 2019, cited above). The dashed and solid lines indicate the difference between dry and lubricated measurements. Friction provided by the plates used during testing can affect cyclic fatigue and strength predictions.

[0655] While the compression test shown in Figures 1A and 1B measures the compressive force across the entire implant or across a large portion of the implant, the "pinch" test, performed as shown in Figure 5, tests the implant's resistance to localized pinching or compression around the implant's periphery. The local resistance indicates the implant's free deformation response and simulates the implant's tactile and apparent "soft" characteristics.

[0656] The results of local lateral compressive force on breast implants are shown in Figure 6 , which shows the relative trends of various implants in the presence and absence of lubricant (dashed lines) considering tactility, palpability, and softness.

[0657] In some embodiments, degradable breast implants comprising a composite scaffold have a shape and mechanical properties that minimize the effects of compressive loads. In some embodiments, degradable breast implants comprising a scaffold have a shape and mechanical properties that minimize the effects of localized compressive loads ("pinching", localized "squeezing"). In some embodiments, degradable breast implants comprising a scaffold have a feel similar to a woman's breast. In some embodiments, degradable breast implants comprising a scaffold have a softness similar to a woman's breast.

[0658] In some embodiments, the degradable implants disclosed herein have projective, diametric, and areal distortions within ±20% of commercially available implants (eg, breast implants).

[0659] In some embodiments, the degradable breast implants disclosed herein have a projective distortion, a diametric distortion, and / or an areal distortion, each independently within ±20% of each characteristic reported for an ideal breast implant in Brandon et al., 2019 (supra). [Example]

[0660] Filler Composition Exemplary soft tissue filler compositions according to some embodiments of the present invention are described below.

[0661] [Table 2]

[0662] An exemplary cross-linked fibrous rh collagen was prepared as follows. Preparation of fibril formation and washing buffers: A fibrillogenesis buffer (FB) stock solution was prepared by adding 1000 mL of double distilled water (DDW) to 23 grams of disodium hydrogen phosphate (SPD) and then filtering the resulting buffer through a 0.22 μm filter. The pH was then adjusted to 11.2 by adding 10 N NaOH, and the resulting buffer was stored at 4°C until use.

[0663] The wash buffer was prepared by mixing 100 mL of FB, 9 mL of HCl, and 891 mL (by weight) of DDW. The pH was adjusted to 7.24 by adding 10 N NaOH, and the resulting buffer was stored at 4°C until use.

[0664] Collagen fibrillogenesis: Nine parts rh collagen (approximately 3.1 mg / mL) was mixed with one part FB, and the mixture was gently stirred at room temperature for 1 hour.

[0665] X-linkages in fibrous collagen: 20 mM EDC was added to the fibrillar rh collagen, and the resulting mixture was gently stirred at room temperature for 2 hours, protected from light.

[0666] The mixture was then centrifuged at 10,000 rpm for 25 minutes at 4°C. The supernatant was discarded, and an equal volume of washing buffer was added. The resulting mixture was shaken and centrifuged. This procedure was repeated three times. The resulting X-linked fibrous rh collagen was stored at 4°C.

[0667] In vitro assay: An aqueous formulation containing X-Fb Coll-EDC20 (5 mg / mL) and a formulation containing X-Fb Coll-EDC20 (5 mg / mL) mixed with HA cross-linked with 20% BDDE (1,4-butanediol diglycidyl) (10 mg / mL) were used.

[0668] The formulation was lyophilized and sterilized with ETO.

[0669] Prior to the experiment, the formulation was reconstituted with 1×DPBS.

[0670] The formulation was homogenized between the two syringes.

[0671] Each formulation (100 μL) was placed into a transwell of a 24-well plate.

[0672] 1 mL of DMEM medium was added to the outside of the well.

[0673] P#8 nHDF cells were used.

[0674] 100 μL of cell suspension (100K cells / mL) was placed on top of each transwell.

[0675] 100 μL of cells (100K cells / mL) seeded on top of the membrane of an empty transwell without formulation was used as a control.

[0676] Plates were incubated at 37°C for 1 or 5 days.

[0677] At each time point, cell viability and proliferation were assessed using the WST assay. Cells were also seeded at various concentrations for a WST assay standard curve.

[0678] The data obtained is shown in Figure 16. As shown, the addition of rh collagen-based filler to the inserts demonstrated beneficial effects on cell proliferation.

[0679] An exemplary process for preparing the rh collagen-derived nanoparticles described herein is as follows.

[0680] The rh collagen is converted to rh gelatin by treating ...

Claims

1. A three-dimensional (3D) biocompatible and degradable soft tissue implant comprising a bioprinted composite scaffold, the composite scaffold being composed of collagen, including cured recombinant human collagen (rh collagen), and a biocompatible synthetic polymer; The composite scaffold comprises: A porous wall; an interior cavity at least partially enclosed within a porous wall; at least one injection port connecting the interior cavity with an outermost surface of the scaffold, the injection port having an opening sized to allow insertion of an injection device therethrough; Including, The soft tissue implant further comprises an injectable matrix within an interior cavity of the scaffold, the matrix comprising an extracellular matrix (ECM) component consisting of rh collagen without curable groups.

2. 2. The soft tissue implant of claim 1, wherein the scaffold further comprises at least one printed vascular network pathway connecting an outermost surface of the scaffold with an internal cavity of the scaffold, the vascular network pathway being sized to allow vascular cells and vascular tissue to penetrate.

3. The soft tissue implant of claim 2 , wherein the scaffold comprises 1 to 1000 printed vascular network pathways.

4. 4. The soft tissue implant of claim 1, wherein the total volume of the internal cavity is between 5 mL and 300 mL, or between 10 mL and 300 mL, or between 50 mL and 300 mL.

5. The soft tissue implant of any one of claims 1 to 4, wherein the curable group comprises a photocurable group.

6. The soft tissue implant of any one of claims 1 to 5, wherein the internal cavity comprises 2 to 30 chambers.

7. The soft tissue implant of claim 6 , wherein at least two of the chambers are interconnected with each other.

8. The soft tissue implant of any one of claims 1 to 7, wherein the scaffold comprises 1 to 15 of the injection ports.

9. 9. The soft tissue implant of any one of claims 1 to 8, wherein the bioprinted composite scaffold is formed by bioprinting a curable composition in a configuration pattern corresponding to the desired shape and dimensions of the soft tissue implant, the curable composition comprising recombinant human collagen (rh collagen) having curable moieties.

10. 10. The soft tissue implant of claim 9, wherein the curable formulation further comprises a biocompatible synthetic polymer having a curable moiety.

11. 11. The soft tissue implant of claim 10, wherein the rh collagen hardenable portion and the synthetic polymer hardenable portion are hardenable when subjected to the same curing conditions.

12. The soft tissue implant of claim 11 , wherein each of the curable portions is a photocurable portion.

13. 13. The soft tissue implant of any one of claims 1 to 12, wherein the biocompatible synthetic polymer comprises polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), poly(N-isopropylacrylamide) (PNIPAAm), poly-4-hydroxybutyrate (P4HB), or a copolymer of any of them.

14. The soft tissue implant of any one of claims 1 to 13, wherein the rh collagen comprises plant-derived recombinant human collagen.

15. 15. The soft tissue implant of any one of claims 1 to 14, wherein the ratio of rh collagen to biocompatible synthetic polymer is from 1:1 to 1:20, or from 1:1 to 1:10, or from 1:2 to 1:

10.

16. The soft tissue implant of any one of claims 1 to 15, wherein the scaffold further comprises at least one extracellular matrix (ECM) component.

17. 17. The soft tissue implant of claim 16, wherein the at least one ECM component has a hardenable portion and the at least one ECM component comprises at least one of hyaluronic acid, fibronectin, heparin, elastin, laminin, and any combination thereof.

18. The soft tissue implant of any one of claims 1 to 17, wherein the scaffold further comprises an integrin binding material.

19. 20. The soft tissue implant of claim 18, wherein the integrin-binding material is an RGD-containing material.

20. 20. The soft tissue implant of claim 18 or 19, wherein the integrin-binding material comprises a curable moiety.

21. The soft tissue implant of any one of claims 1 to 20, wherein the matrix further comprises cells.

22. 2. The soft tissue implant of claim 1, wherein the extracellular matrix (ECM) component consists of EDC-crosslinked fibrillar rh collagen.

23. The soft tissue implant of any one of claims 1 to 22, wherein the matrix further comprises an integrin binding material.

24. 22. The soft tissue implant of claim 21, wherein the cells comprise pericytes, adipose-derived stem cells, preadipocytes, endothelial cells, progenitor cells, hematopoietic cells, adipocytes, or any combination thereof.

25. 22. The soft tissue implant of claim 21, wherein the cells comprise stromal vascular fraction (SVF) isolated from adipose tissue.

26. The soft tissue implant of any one of claims 21 to 24, wherein the cells comprise a minimally processed extract from adipose tissue.

27. The soft tissue implant according to any one of claims 21 to 26, wherein the volume of the matrix is ​​between 5 ml and 300 ml.

28. 28. The soft tissue implant of any one of claims 1 to 27, which is a breast implant.

29. 28. The soft tissue implant of any one of claims 1 to 27, wherein the soft tissue is selected from breast tissue, facial tissue, neck tissue, muscle tissue, joint tissue, jaw tissue, buttock tissue, hand tissue and chest tissue.

30. 10. A method of preparing an implant according to claim 1, comprising sequentially forming multiple layers in a structural pattern of the scaffold, at least a portion of the layer is formed from a formulation comprising the recombinant human collagen having at least one curable group, a synthetic polymer having at least one curable group, and optionally an ECM component having a curable group and / or an integrin-binding material having at least one curable material; The method optionally further comprising injecting a matrix into at least the internal cavity of the scaffold via an injection port.

31. The method of claim 30 , wherein the curable group comprises a photocurable group.

32. 32. The method of claim 31 , wherein the photocurable group comprises an acrylic or methacrylic group.

33. A method for producing a scaffold comprising: at least one formulation for forming a scaffold, the at least one formulation comprising curable recombinant human collagen (rh collagen) and a biocompatible synthetic polymer, the biocompatible synthetic polymer optionally having curable groups; and an injectable matrix comprising an ECM component for forming the scaffold matrix, the ECM component being comprised of rh collagen having no curable groups; A kit comprising: Kit for the preparation of bioprinted soft tissue implants.

34. 34. The kit of claim 33, further comprising cells.

35. A soft tissue implant according to any one of claims 1 to 29 for implementation of soft tissue augmentation and / or reconstruction and / or regeneration in a subject in need thereof.

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