Particulate materials for tissue mimics

Abstract

Two-component biomaterials and methods that replicate both the structural complexity and diverse molecular composition necessary to generate tissue form and function. It is an object of the present invention to use the unique combination materials and methods herein to provide pharmaceutical compositions, medical devices, tissue regeneration scaffolds, and scaffolds for 3D tissue culture (tissue on a chip, lab-grown meat, stem cell differentiation for research) that contain significant amounts of acellular tissue particles that are densely loaded and held together via crosslinks between the acellular particles and thiolated proteins.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 125,280, filed on December 14, 2020; U.S. Provisional Patent Application No. 63 / 263,228, filed on October 28, 2021; and U.S. Provisional Patent Application No. 63 / 264,564, filed on November 24, 2021.

[0002] The present invention relates to methods and devices for tissue engineering. More specifically, the present invention relates to extracellular matrix tissue particle - based biomaterials and bioinks that can be used to generate three - dimensional printed layered tissues such as articular cartilage scaffolds.

Background Art

[0003] The extracellular matrix of biological tissue is a dynamic environment that provides macro - scale structure and protection for cells under physiological loads while also providing micro - scale cell contacts for growth, differentiation, and receptor signaling. Any specific structure and composition of both healthy and engineered extracellular matrices can determine mechanical behavior and induce cell responses.

[0004] Biological tissues provide basic load - bearing functions throughout the body. There are many remaining challenges in regenerative medicine. For example, some tissues such as articular cartilage have demonstrated that repair after injury is impossible [Mow, V.C., Ateshian, G.A. & Spilker, R.L. Biomechanics of diarthrodial joints: a review of twenty years of progress. J Biomech Eng 115, 460 - 467 (1993)]. Tissues with high extracellular matrix content and low cell density require unique tissue engineering designs and approaches for robust and integrated repair solutions.

[0005] Healthy biological tissues are often composed of heterogeneous and distinct tissue layers. For example, in articular cartilage, each layer has a unique ratio of the two major extracellular membrane proteins, collagen and aggrecan. Each layer also has a different cell density. The composition of each cartilage layer and the interactions between the layers allow articular cartilage to provide excellent joint lubrication and facilitate the transmission of load during normal movement. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Despite decades of innovation, the successful replication of biological tissues using tissue-engineered constructs to repair post-injury defects remains a challenging task. To enable efficient and effective repair of tissue defects, there is a significant need to engineer replication scaffolds that mimic the structural properties of tissues and present natural biochemical cues to promote cell growth and integration [Camarero-Espinosa, S., Rothen-Rutishauser, B., Weder, C. & Foster, EJ Directed cell growth in multi-zonal scaffolds for cartilage tissue engineering. Biomaterials 74, 42-52 (2016)]. [Means for solving the problem]

[0007] The present invention relates, in general terms, to a biomaterial comprising (1) a decellularized and particulate extracellular matrix and (2) a thiolated polymer, as well as to methods for the manufacture and use of the biomaterial. Specifically, in one embodiment, the present invention relates to treating a diseased or damaged area of ​​tissue by combining two biomaterial components and injecting the combined material into the damaged area. The present invention can be used as a biomaterial for medical implants / procedures or three-dimensional cell cultures and / or three-dimensional tissue prints. Methods for manufacturing the individual components and the combined biomaterials thereof are also provided herein.

[0008] Many tissues are layered with specific and complex heterogeneous structures to provide their critical functionalities. Many manipulated constructs for in vivo tissue repair, 3D printing, organoid fabrication, chip-based laboratories, in vitro analysis, and laboratory-grown meat fail to replicate the mechanical properties, unique layering, or complexity of tissues, often relying on exogenous compounds to crosslink materials to a stable scaffold. To address the need for mechanically robust, complex, and layered scaffolds, extracellular matrix (ECM) tissue particle-based biomaterial inks have been developed that can be layered or used alone to produce reliable tissue mimics for 3D organ culture or implantable medical devices for tissue repair, readily extruded and polymerized into mechanically robust constructs via disulfide bonds. pECM bioinks, which are ECM-based particle bioinks, utilize functionalized macromolecules that are combined with or loaded with cell-free tissue particles. When combined, these two materials cross-link via disulfide bonds to produce a stable tissue mimic or to repair a scaffold.

[0009] In particular, the present invention relates to a composite material of functionalized macromolecules that can be crosslinked with cell-free tissue particles by disulfide bonds. These functionalized macromolecules may be natural or synthetic. The tissue particles may be obtained from the tissues or organs of any animal or human. The obtained tissue particles may be mechanically disrupted and cell-free, and then functionalized or combined with functionalized macromolecules. The present invention also relates to a method for combining composite materials and using this mixture to produce tissue mimics. There are many uses for the biomaterials disclosed herein, including applications for clinical tissue repair, such as organoid development using biomaterials mixed with living cells, as a commercially available 3D printable biomaterial ink that can be used sequentially to produce layered tissue mimics, as an injectable / implantable medical implant (e.g., as a reparative material for cartilage, skin, muscle, bone, kidney, liver, and heart), as a chip-on tissue / laboratory site (of any tissue type) for testing of medical procedures, and as an organoid development using biomaterials mixed with living cells, as well as applications by laboratories / companies to answer various problems requiring ECM-based cultures or 3D cultures, tissue systems (i.e., to function as models for tissue, disease, regeneration, etc.), and finally as a matrix for producing laboratory-grown meat.

[0010] For a more detailed understanding of the present invention, the following detailed description shall be made with reference to the accompanying drawings. [Brief explanation of the drawing]

[0011] [Figure 1]This is a set of images and two graphs (labeled (A)-(D)) showing a natural tissue-based cell-free biomaterial crosslinking ECM particles to a functionalized macromolecule (shown here as thiolated hyaluronan) via disulfide bonds. (A) Scaffold crosslinking by the formation of disulfide bonds between the thiolated moiety on the macromolecule, thiolated hyaluronan in this example, and the ring-opening sulfahydryl group on the tissue particle, thereby forming a stable three-dimensional network. (B) By comparison with a known amount of cysteine, it was measured via the Ellmann method that the particles contain sulfahydryl groups to interact with thiolated hyaluronan. (C) Addition of either or both of the particles, PEGDA, led to the initiation of crosslinking and polymerization within 30 minutes, with further polymerization occurring for about 2 hours. In a control containing only thiolated hyaluronan, it remained liquid after 2 hours, demonstrating the role that the particles play in increasing the crosslinking rate. (D) TCEP buffer is a reducing agent that breaks down disulfide bonds. Incubation in TCEP buffer ensures that the hyaluronan (HA)+ particle gel crosslinks via disulfide bonds. [Figure 2] This is a set of eight images. Heprasil and Gelin-S are additional types of functionalized macromolecules (in addition to thiolated hyaluronic acid / hyaluronan shown, for example, in Figure 1). Heprasil is a thiol-modified heparin / hyaluronic acid mixture, and Gelin-S is thiolated gelatin. When combined with cell-free microparticles (i.e., fat, dermis, cartilage), all functionalized macromolecules cross-link with the cell-free microparticles to form a stable and robust scaffold. [Figure 3] This is a set of images demonstrating that extracellular matrix can originate from a wide variety of tissue types, which can then be decellularized and granulated for use as ECM particles. Once granular, the tissues are all combined with functionalized macromolecules (in this example, all tissues are functionalized with thiol groups), mixed, and can be dispensed through any needle / nozzle size (the limiting factor is the maximum particle size that can be embedded in the ink). [Figure 4]This is a set of diagrams illustrating the use of ECM particle biomaterial as a bioink using layered printing for the production of cartilage mimics with mechanically robust structure and lubricity. (A) In this illustrated method, cartilage tissue is decellularized and made into particles of 40-100 μm. The resulting particles are encapsulated in hyaluronan and printed to produce a mechanically robust cartilage scaffold. The top lubricating layer, consisting solely of hyaluronan, is printed through a second nozzle to replicate the lubricating surface features of cartilage. (B) The scaffold is printed at two different densities using a 22-gauge printhead tip to demonstrate the role of macropores of different sizes in nutrient dispersion, cell viability, and mechanical properties. For comparison purposes, a third group can be provided by casting the scaffold rather than printing it. (C) The lubricating layer can be printed as a surface layer on a cartilage tissue construct, in which the surface layer consists of cross-linked hyaluronan, a polysaccharide that plays an important role in the lubricity of conventional articular cartilage. [Figure 5]This is a set of figures, images, and graphs demonstrating that when biomaterial inks are used for 3D printing, layered tissues can be generated using printing, and that tissue-specific layering can be generated by specifically using unique cell-free tissue inks. When biomaterial inks are printed on articular cartilage via extrusion, the ink can be used to generate mechanically robust intermediate and deep regions with smooth, low-friction surface regions. (A) Incorporation of microtissue particles into hyaluronan ink enables printing of a wide range of structures. Here, scaffolds were generated with a range of macropores, i.e., 1 mm, approximately 400 μm, and no pores. (B) The addition of small macropores had no effect on the overall compressibility coefficient of the scaffold, measured under unrestricted compression using a 10 mm flat pressure plate. (C) The incorporation of pores did not change the expansion level of the structure, ensuring that this expansion was largely due to the chemical crosslinking of the ink rather than the shape of the structure. Printing the surface areas of (D and E)HA significantly reduced surface roughness, friction, and adhesion (surface measured with respect to AFM using a 2 μm spherical probe). * represents p < 0.05, ** represents p < 0.001, and Tukey's true significance test is performed following a two-way ANOVA. The scale bar in (A) is 1 mm. [Figure 6] This set of figures, images, and graphs demonstrates that when biomaterial inks are printed in 3D, the printing process can be used to introduce tissue scaffolds with macropores. Increasing the porosity of the constructs led to improved cell distribution and viability. (A, B) Chondrocytes were extracted from bovine knee joints, stained with a fluorescence-enhancing dye (CFSE), and seeded at the top of all constructs. The percentage of dead cells was measured by quantification of cells stained with CFSE (live) or ethidium homodimer (dead) at 7 and 14 days after seeding. (C, D) The distribution of chondrocytes throughout the scaffold was measured by quantification of CFSE-stained cells at three tissue depths: superficial, middle, and deep layers after 7 days of culture. * indicates p<0.05, followed by Tukey's true significance test using two-way ANOVA. [Figure 7]This is a set of diagrams and images demonstrating that biomaterials can be designed for in-situ use to provide three-dimensional scaffolds for studying tissue regeneration, repair, or barrier interaction / integration. The biomaterial is designed and printed to be loaded into an example of cartilage showing a specific tissue defect, and after 7 days, the printed construct can be incorporated into and adhere to the surrounding intrinsic tissue. (A, B) The custom-made tissue defect is measured and entered into CAD software to generate a custom-designed construct that fits into the defect. The custom-made construct is generated using extrusion printing and immediately introduced into the defect for polymerization. (C) After 7 days, the construct is bisected and imaged for direct observation of the integrity of the scaffold and its integration with the intrinsic tissue. The scaffold adheres to the intrinsic tissue even while force is being exerted during bisecting. In addition, CFSE-stained cells (grayscale green-grayish white) introduced after polymerization accumulate where the intrinsic tissue and scaffold match. [Figure 8] This is a set of figures and images demonstrating that 3D printing of ECM particle bioink is adjustable for a wide range of shapes and sizes, demonstrating the potential for clinically demand-driven applications. (A) For clinical applications, it is essential to develop materials that can be applied to patient-specific defects that vary in size and degree in localized cartilage damage within complex joints. (B, C) It is demonstrated that using ECM particle bioink, a customized defect shape can be measured, applied to a 3D CAD model, and printed via extrusion to produce a multilayer construct suitable for implantation. Here, the design of a two-layer cartilage construct using tissue particles for enhanced tissue structure in addition to an engineered lubricating layer can be immediately printed and applied to repair joint defects. [Figure 9]A pair of graphs demonstrates that ECM particle bioinks can be evaluated for printability in many ways. Figure 9 shows the polymerization time (A) and shear viscosity reduction behavior (B) of ECM particle bioinks when using cartilage tissue of size 40–100 μm and 20% thiolated hyaluronan. Rheological properties were measured using a flow procedure with a steady-state sulfur step set on a rheometer (AR-G2, TA Instruments). Experiments were performed using parallel plates consisting of 8 mm diameter plates with a gap distance of 800 μm and bearing stiffness mode at room temperature. Shear sweeps were measured as a function of shear velocity 0.01–200 s⁻¹, no normal force, and pre-shear velocity of 10 for 10 seconds and 0 velocity. Time sweeps were performed using parallel plates set at 37°C. [Figure 10] See Figure 9 for a pair of graphs (Figures 10(A) and 10(B)), along with two additional graphs (Figure 10(C) showing the polymerization coefficient of the ECM particle bioink scaffold and the time it takes to reach a crosslinked state (where the graph flattens) and Figure 10(D) showing imaging of ink extrusion out of a 22 gauge nozzle used for 3D printing to demonstrate the smoothness and consistency of the extruded ink). [Modes for carrying out the invention]

[0012] Successful bioscaffolds function as structural supports for both cell integration and mechanical integrity. Ideally, an implantable bioscaffold is a substrate for cell adhesion and proliferation, allows for sufficient diffusion of nutrients and waste through the scaffold, mimics the structural heterogeneity of the original tissue, and mechanically responds to the normal load applied to the manifested tissue. Therefore, the extracellular matrix (ECM) of decellularized tissue is a very favorable scaffold for tissue repair because the ECM provides the same structural support as the original tissue, while additionally presenting well-known physiological cues that enable cell invasion and integration into the scaffold from surrounding tissue (in vivo) or from introduced cells (in vitro).

[0013] However, in practice, the application of whole intact decellularized tissue for tissue defect repair is limited by morphological constraints and may be limited by high conventional tissue density (depending on the tissue that needs to be replicated). The extracellular matrix (ECM) of decellularized donor tissue can be broken down into smaller particles and then fabricated into composite materials by densely loading these particles in hyaluronic acid / PEGDA crosslinked hydrogels to generate regenerative models for cartilage repair. Dense particle loading restores the mechanical properties of larger decellularized tissues and significantly improves cell infiltration into the scaffold. While ECM particle-loaded hydrogels represent a promising advance in scaffold technology, it is crucial to produce scaffolds that also mimic tissues with different layered structures (e.g., articular cartilage) by utilizing the chemical characteristics of crosslinking without Michael addition between functionalized hyaluronic acid and PEGDA diacrylate groups.

[0014] This invention addresses the problems of conventional bio-scaffolds by generating scaffolds that directly utilize cross-linking interactions between functionalized macromolecules (natural or synthetic) and tissue particles.

[0015] Advanced manufacturing technologies, particularly 3D printing, offer several advantages for tissue engineering and bio-scaffolding. A wide range of applications utilize extrusion-based printing, which allows for size synchronization, the use of a broad range of printable materials, the ability to impose constraints through unique layers, and the printing of bespoke shapes and sizes. Conventional attempts have included extrusion-based printing of conventional synthetic polymers to produce tissue-engineered scaffolds because these polymers offer enhanced engineering control. Unfortunately, synthetic polymers alone generally fail to perfectly mimic the structure or function of most natural tissues. Instead, natural polymers, often composed of proteins abundant in the body such as hyaluronane, arginate, and collagen, have been used in tissue applications involving engineered 3D printing. Unfortunately, most natural polymer-based hydrogels rely on ultraviolet light and other further photopolymerization initiators to crosslink and form stable scaffolds, and do not additionally replicate the complex protein-polysaccharide interactions found in most conventional tissues. To increase the complexity of polymers in natural hydrogels, researchers have developed extracellular matrix-based bioinks by digesting decellularized tissues into a liquid (monomer) state. [JSChoi,HJYang,BSKim,JDKim,JYKim,B.Yoo,K.Park,HYLee,YWCho,Journal of Controlled Release 2009,139,2]. In these procedures, proteases digest the tissue, breaking down its robust ECM structure and thereby liquefying it. Unfortunately, tissue digestion also disrupts the protein organization that provides the tissue with essential components such as its unique structure, ECM architecture, protein interactions, and cell attachment sites, which would otherwise create an ideal regenerative tissue environment.

[0016] This specification describes the development of cell-free tissue-based biomaterial material compositions that can be injected into defective or damaged areas in patients (human or animal) or used as 3D printed biomaterial resins (pECM bioinks) to generate robust tissue mimics as architectures for 3D organ cultures or tissue application on chips. In one embodiment, the biomaterial is derived from purely tissue-based and natural extracellular matrix components. In other embodiments, the natural tissue matrix is ​​crosslinked with a functionalized synthetic polymer. The methods and material compositions taught herein affect sulfhydryl groups on cysteine ​​in the decellularized tissue to crosslink with functionalized macromolecules, thereby generating disulfide bonds between the two biomaterial ink components. Unlike biomaterial inks formed from complete tissue digestion, cell-free microparticles are produced via mechanical grinding, resulting in ECM microparticles that retain the micromechanical and biological properties of the original tissue. The ground particles are densely loaded in a hydrogel solution of functionalized macromolecules and can be injected / applied to either shape or printed via extrusion. Using techniques employed in applications such as printing viscous clay, we have developed a printed ECM particle biomaterial ink. We have demonstrated that printing with the ECM particle biomaterial ink enables the creation of layered tissues and the printing of macropores in tissue mimics. Furthermore, we demonstrate the simplicity of printing and implanting custom-made scaffolds for patients using layered, lubricated articular cartilage scaffolds that are printed to a specific shape and implanted within the defect.

[0017] The present invention provides tissue extracellular matrix particles obtained from animal or human tissue or organs. The extracellular matrix particles are mechanically micronized to a selected size. In various embodiments, the particles can be 0 - 100 μm, 100 - 250 μm or 250 - 500 μm. The particles are decellularized using chemical means to remove the cellular components of the tissue, lyophilized, and size - sorted into different size classifications. The decellularized extracellular matrix particles have native growth factors, attachment sites for cells, and a complex tissue - specific architecture that will promote cell interaction and regeneration cascades.

[0018] The particles are combined with a polymer (natural or synthetic, preferably natural) functionalized by thiolation (due to having open thiol (-SH) groups) and dissolved for easy mixing with the particles. In different embodiments, the percentage of molecular groups on the macromolecule degraded to have functionalized or open thiol groups can fall within one of the following categories: 0 - 15%, 15 - 30% and / or 30 - 60%. The percentage of functionalization indicates the cross - link density of the combined material, where a higher cross - link level results in a smaller pore size / lower material porosity and a more robust material.

Example

[0019] Example 1 - Materials and Methods - Particulate tissue extracellular matrix cross - linked with thiolated polymer generates scaffolds Origin of tissue ECM: Tissue ECM is most often obtained from animals or humans after terminal treatment, but can also be obtained during procedures involving tissue replacement or tissue extraction (e.g., liposuction). The tissue is harvested fresh (within 48 hours of slaughter) or supplied as frozen tissue (instantly frozen within 48 hours of slaughter or harvest). The tissue is retrieved from the target tissue using a scalpel and / or dissecting scissors. It is essential not to harvest tissue in areas that meet different types of tissue (at the ends) to ensure that only the desired tissue type is harvested. After harvest, the tissue is about 1 cm2 After being cut into pieces, the tissue is flash-frozen and preserved or pulverized. The tissue may be of heterogeneous, homogeneous, or autologous origin and may be preserved as a single-source tissue (for example, for individualized administration or autografts) or pooled with other samples of the same tissue type for more versatile applications.

[0020] Dismantling the organization: The tissue is ground in two stages of the preparation method: Stage 1. after extraction from the tissue or organ (but still consisting of cellular contents) and Stage 2. after decellularization and freeze-drying. In Stage 1, the flash-frozen tissue fragments are confined in a flash-frozen stainless steel grinding jar equipped with freely floating stainless steel spheres. The grinding jar containing the tissue is ground at high speed vibration (e.g., 30 revolutions / second with a TissueLyser II grinding device) for 30 seconds to 1 minute. In Stage 2, the decellularized, freeze-dried acellular cartilage tissue particles are further ground in a stainless steel grinding jar (room temperature grinding jar and tissue) at the same speed (e.g., 30 revolutions / second with a TissueLyser II grinding device) for 60 seconds. After the second grinding, the particles are sized to different sizes, namely <40 μm, 40-100 μm, 100-250 μm, and 250-500 μm, using a vibrating sieve (e.g., supplied by Endecott).

[0021] Decellularization: Microparticles are decellularized using a series of chemical and enzymatic methods. After the first bulk tissue pulverization, cells are degraded and detached from the tissue by initial in-liquid treatment in 2% sodium dodecyl sulfate (SDS) at 37°C with stirring for 8 hours. Then, the SDS is removed by rinsing overnight in a 1× phosphate buffer solution. Optionally, the tissue particles can then be further degraded of residual cellular and genetic material by enzymatic treatment (e.g., 0.1% DNase for 3 hours) at 37°C with gentle stirring. The microparticles are rinsed in PBS, flash-frozen in liquid nitrogen, lyophilized, and cryopreserved at -80°C until the second pulverization (detailed earlier). The extracellular matrix is ​​preserved substantially without cells and can be described as an aggregate of small particles. These particles can be used for research purposes in combination with functional macromolecules, or they can be sterilized, stored, and fabricated for implants.

[0022] Polymer functionalization: Many polymers can be functionalized to have open -SH groups that can form disulfide bonds with -SH groups on the tissue ECM. One example of macromolecular functionalization is the thiolation of hyaluronic acid molecules. In this example, the glucocorate carboxyl group on hyaluronan (HA) was replaced with a thiol group according to an established protocol [D.Eng, M.Caplan, M.Preul, A.Panitch, Acta Biomaterialia 2010, 6, 2407]. Briefly, hyaluronan (molecular weight 100 kDa, Lifecore Biomedical) was dissolved at 10 mg / ml in degassed Milli-Q water. Dithiobispropanoate dihydrazide (DTP) (Frontier Scientific) was added to this solution to lower the pH to 4.5-4.75, and then (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (ThermoFisher) was added to initiate the reaction. The pH was maintained at 4.5-5 for 50 minutes. After 50 minutes, the reaction was stopped by raising the pH above 7. Next, dithiothreitol (DTT) (Fisher Scientific) was added to raise the pH above 8.5, and the solution was stirred at room temperature for 24 hours. After 24 hours, the pH of the solution was lowered, and the liquid was passed through a dialysis tube (10kDa membrane cutoff, Spectrum The solution was transferred to Labs and dialyzed with eight solution exchanges in HCl solution supplemented with 100 mM sodium chloride, followed by four solution exchanges in HCl solution without supplementation. Dialysis was completed in a sealed chamber while continuously blowing nitrogen gas into the HCl solution. Using each batch of HA thiolation, the substitution rate can be confirmed to be within the specified inventive range of 0-15%, 15-30%, or 30-60% (thiolated mmol / non-thiolated mmol) using a standard Elman assay according to the manufacturer's protocol (Elman solution, ThermoFisher).

[0023] Combine with functionalized polymers: Size-sorted and lyophilized cell-free tissue particles are combined with a functionalized polymer (either purchased or prepared as described above, and either natural or synthetic) to produce a biomaterial composition. The lyophilized and functionalized polymer (see above) readily dissolves in 1×PBS, dissolving to produce an aqueous solution at twice the desired polymer concentration (e.g., 20 mg / ml macromolecules), which is neutralized to pH 7 and loaded into a syringe, carefully removing any bubbles formed during syringe loading. In a separate syringe, the size-sorted cell-free tissue particles are mixed with 1×PBS at twice the desired final ratio (e.g., 0.4 g / ml). When ready to be combined (e.g., for device delivery into defects, tissue bioprinting, or 3D cell culture), the syringes are joined together using a custom-made Luer-lock connector with a crosshair across the opening to facilitate mixing. The solution is syringed approximately 30 times until mixing is complete and the material flows smoothly through the Luer-lock mixing connector. The final mixture will have a macromolecular concentration approximately half that of each individual component (throughout this example, this would be 10 mg / ml, and the particle concentration would be 0.2 g / ml).

[0024] Example 2 - Therapeutic and Research and Development Uses The present invention provides cell-free tissue particles (derived from and combined from one or more tissues) crosslinked to a thiolated polymer, for repairing and providing a regenerative scaffold for missing, damaged, diseased, or lost tissue or components of organs in a subject requiring repair (either an animal or human subject). In certain embodiments, the material composition may be used to treat missing or damaged organs, including but not limited to articular cartilage, muscle, skin, ligaments, subcutaneous fat beneath the skin, kidneys, and liver.

[0025] In particular, biomaterial compositions may be used to treat diseases, conditions, or injuries that involve the loss of bulk tissue and which can benefit from a structurally mature regenerative tissue scaffold that immediately fills a volume, provides mechanical function to the damaged tissue, and allows cells to migrate, adhere to, and proliferate in a regeneration cascade. Such diseases may include, but are not limited to, facial wasting syndrome, articular cartilage damage due to trauma, osteoarthritis, ulcers, large or deep burns and / or wounds, breast reconstruction, bulk muscle loss, and vocal cord fold disorders.

[0026] In certain embodiments, the material composition may be used as a biomaterial ink extruded using 3D printing technology. Applications of using this material for 3D printing include, but are not limited to, tissues on chips, tissue mimics, 3D cell cultures, personalized drug administration, personalized treatment testing, drug toxicity testing, and barrier tissue crosstalk studies.

[0027] Example 3 - Medical Device Biomaterial compositions may be formulated as medical devices for injection and / or permanent implantation into a subject. These medical devices would use solubilized polymers as carriers for extracellular matrix tissue particles to deliver material to the required area. More specifically, biomaterial compositions would be applied to a defect or injury area to promote regeneration and / or provide structural stability in the damaged area.

[0028] In some embodiments, this polymer can also be mixed with extracellular matrix particles and, prior to injection into the desired site, with external growth factors, region-specific anti-inflammatory agents, or other required active and FDA-approved pharmaceuticals (e.g., TGFβ, IGF-I, PRP, corticosteroids, etc.). The components of the composition of the present invention can be supplied individually, packaged, and mixed before administration.

[0029] The thiolated polymers used to constitute the biomaterial composition shall be biocompatible and shall avoid irritation or systemic reaction to conventional tissues / hosts. Furthermore, if desired, multiple functionalized polymers (e.g., hyaluronan and collagen) may be combined with extracellular matrix particles. In preferred embodiments, a single type of functionalized polymer is used.

[0030] Example 4 - Tissue regeneration scaffold One important aspect and application of the present invention is the use of crosslinked extracellular matrix particles combined with thiolated polymers to generate an ideal scaffold for tissue regeneration. In a favorable embodiment, the scaffold is injected into a region of the body, crosslinking and adhering to the surrounding conventional tissue via activated functional groups on macromolecules. Growth factor residues in the extracellular matrix particles encourage cell transport and adhesion. As cells in the tissue and surrounding tissue proliferate and / or begin to lay new extracellular matrix, the gaps between the tissue particles are filled with newly grown tissue. In another embodiment, the biomaterial compositions described herein can be formed under in vitro sterile conditions. Cells can be seeded in the scaffold slurry before or after crosslinking. The cell scaffold can be maintained and cultured until it reaches a desired tissue maturity, in which case the regenerative scaffold can be implanted in the patient (for example, when the procedure progresses to a matrix-assisted autologous chondrocyte implantation procedure in which the tissue or cells must be cultured outside the patient's body).

[0031] Example 5 - Cell Culture Substrate Many cells require or exhibit enhanced growth on substrates other than tissue culture plastics to grow outside their innate tissue environment and maintain proper function. In particular, cells that depend on pericellular and external signaling for differentiation require non-plastic materials (e.g., stem cells and pluripotent cells). In certain embodiments, the biomaterial compositions taught herein can be used for cell culture of various cells. The particle tissue source would require a specific extracellular matrix environment for cell culture that potentially provides signals to differentiate stem cells or maintain cells in a tissue-specific phenotype. In certain embodiments, cells can be seeded on the surface of the crosslinked biomaterial invention described herein. In other embodiments, these cells can be encapsulated in a thiolated polymer, mixed with particles as described, and seeded in a template for a three-dimensional culture environment.

[0032] Example 6 - Printing for individualized drug delivery or 3D cell culture CAD designs for specific defect shapes or desired 3D culture models shall be loaded into the 3D printer, and their use shall be adjusted to the application by adjusting the infill density. Freshly mixed biomaterial inks may be loaded into the print cartridge, and printing shall be initiated within 5-10 minutes of mixing. The inks shall be maintained at 4°C to prevent pre-crosslinking, and printing shall be performed within 30 minutes.

[0033] Example 7 - Scaffold for laboratory-grown meat substitute In one embodiment, the material composition may be used as a tissue-specific substrate for artificially growing meat or meat substitutes in the laboratory. This material can be used to differentiate cells into tissue-specific cells (e.g., muscle), and as such, the scaffold facilitates the production and capability of a novel tissue matrix for manufacturing meat substitutes.

[0034] Example 8 (Part 1) - Cartilage scaffold having hyaluronic acid crosslinked to hyaluronic acid cartilage particles Decellularized, fragmented, and lyophilized cartilage microparticles were encapsulated in a thiolated hyaluronic acid (HA) hydrogel at a ratio of 0.2 g / ml via a mixture of two groups: 1. lyophilized particles in PBS and 2. functionalized HA dissolved in PBS and neutralized to pH 7. The HA was already thiolated to 22% and readily dissolved upon introduction into sterile DPBS. This hydrogel was incubated at 37°C for 30 minutes to facilitate disulfide bond crosslinking between the ring-opening sulfide groups on the cysteine ​​of the particles and the thiol groups on the functionalized HA molecules, forming a stable three-dimensional structure. The incubation temperature can be varied around the roughly optimal temperature of 37°C (e.g., 32°C to 42°C). Lowering the temperature, for example to room temperature, may be necessary to extend the incubation time.

[0035] Example 8 (Part 2) - Proximal Renal Tubular Tissue Mimic In another example, to print a model of the proximal renal tubules, a freshly prepared kidney pECM bioink (e.g., containing thiolated HA) with a particle size <100 μm was prepared using kidneys from a porcine source. This ink was printed using direct ink writing on a sterile multi-nozzle extrusion printer (BioX, Cell Ink Life Sciences) in parallel with the carcass ink used to print the tubular channels. The ECM particle bioink was extruded into a custom-made PDMS mold using a pneumatic print cartridge and a 22-gauge nozzle. The ink was extruded at a speed of 5 mm / second at a pressure of 40–60 kPa (adjusted in real time to ensure interlayer print consistency). The ink was set to pre-flow and post-flow 30 milliseconds before and after the print stage. The nozzles were calibrated prior to printing to ensure alignment of multiple printheads and specific inks. After printing and crosslinking, the scaffold was cooled to liquefy the carcass ink, and this liquid was removed using reflux.

[0036] Example 8 (Part 3) - Layered bone-cartilage printed in vitro using 3D printing In a further example, osteochondral (bone and articular cartilage) scaffolds were printed for custom knee defect fillings following the procedure below. pECM bioink (e.g., decellularized cartilage particles in PBS solution mixed with thiolated HA solution) using freshly prepared extracellular matrix of either bone or cartilage was printed using direct ink writing on a sterile multi-nozzle extrusion printer (BioX, Cell Ink Life Sciences). A 3D print design was generated in open-source CAD software (OnShape) according to measurements of the defect in the injured knee and transferred to the 3D printer for segmentation. The ECM particle bioink was extruded using a pneumatic print cartridge and 22-gauge nozzle ink. The ink was extruded at a speed of 2 mm / second and a pressure of 70-90 kPa (adjusted in real time to ensure interlayer print consistency). The ink was set to pre-flow and post-flow for 30 milliseconds before and after the print stage. The ink was printed with a grid pattern at 75% infill. For the topmost lubrication layer, surface area ink was printed at a pressure of 3–10 kPa and a speed of 20 mm / second, without pre-flow or post-flow. The nozzles were calibrated prior to printing to ensure alignment of multiple printheads and specific inks. Immediately after printing, the construct was inserted into the defect area to facilitate post-embedding polymerization, a crucial process for the integration of the existing tissue. See, for example, Figure 8C.

[0037] Example 8 (Part 4) - 3D Printed Layered Scaffold in Situ In another example, following the same procedure as in Part 3 of Example 8, the print was directly placed into the knee defect and polymerized within the joint (37°C, 30 minutes, 18 minutes until crosslinked based on rheology, but with an incubation period of 30 minutes to allow for slight variations in the environment).

[0038] Example 8 (Part 5) - Dermal scaffold with gelatin cross-linked to dermal particles In another example, porcine dermal tissue was ground into small pieces, decellularized, subdivided, and freeze-dried. The decellularized particles were combined with Gelin-S (Advanced Biomatrix), a form of thiolated gelatin, at a ratio of 0.2 g / ml in two groups: 1. freeze-dried particles in PBS and 2. Gelin-S dissolved in PBS and neutralized to pH 7. The hydrogel was incubated at 37°C for 30 minutes to facilitate disulfide crosslinking between the ring-opening sulfide groups on the cysteine ​​of the particles and the thiol groups on the gelatin molecules, forming a stable three-dimensional structure composed of dermal ECM and type I collagen, along with extracellular matrix components that would prepare healthy skin after wound healing.

[0039] Example 8 (Part 6) - Bone or cartilage scaffold containing heparin sulfate In another example, porcine bone and hyaline cartilage tissue were ground into small pieces, decellularized, subdivided, and lyophilized. The decellularized particles were combined with Heprasil (Advanced Biomatrix), a combination of thiolated heparin sulfate and thiolated hyaluronan, at a ratio of 0.2 g / ml in two groups: 1. lyophilized particles in PBS and 2. Heprasil dissolved in PBS and neutralized to pH 7. The hydrogel was incubated at 37°C for 30 minutes to facilitate disulfide bond crosslinking between the ring-opened sulfide groups on the cysteine ​​of the particles and the open thiol groups in the Heprasil solution. Scaffolds were generated in this manner for tissue engineering applications of both bone and hyaline cartilage. The particle-filled solution can be easily applied to defects in either bone or cartilage (using decellularized bone or cartilage tissue, respectively), creating an environment for osteogenesis or chondrogenesis of surrounding innate pluripotent cells.

[0040] The present invention provides compositions of material components used in generating tissue mimics and combined materials / tissue mimics. Furthermore, the present invention provides methods for combining and forming composition materials, and methods for using combined materials to mimic tissue in various application cases, including but not limited to pharmaceuticals, medical devices, 3D printing of biomaterial inks, and tissue-specific organ cultures.

[0041] References: A.-V.Do,B.Khorsand,SMGeary,AKSalem,Advanced healthcare materials 2015,4,1742. BNBrown, SF Badylak, Translational Research 2014, 163, 268. T. Novak, B. Seelbinder, CMTwitchell, SLVoytik‐Harbin, CPNeu, Advanced Functional Materials 2016, 26, 5427. JEBarthold, BMst. Martin, SLSridhar, F. Vernerey, SESchneider, A. Wacquez, VLFerguson, S. Calve, CPNeu, Advanced Functional Materials 2021,2103355. NASears,DRSeshadri,PSDhavalikar,E.Cosgriff-Hernandez,Tissue Engineering Part B:Reviews 2016,22,298. EAAisenbrey, A. Tomaschke, E. Kleinjan, A. Muralidharan, C. Pascual-Garrido, RRMcLeod, VLFerguson, SJ Bryant, Macromolecular Bioscience 2018,18,1700267. Y. Lai, H. Cao, X. Wang, S. Chen, M. Zhang, N. Wang, Z. Yao, Y. Dai, X. Xie, P. Zhang, X. Yao, L. Qin, Biomaterials 2018,153,1. KCHung, CSTseng, LGDai, S.hui Hsu, Biomaterials 2016,83,156. X. Yang, Z. Lu, H. Wu, W. Li, L. Zheng, J. Zhao, Materials Science and Engineering C 2018,83,195. JSChoi,HJYang,BSKim,JDKim,JYKim,B.Yoo,K.Park,HYLee,YWCho,Journal of Controlled Release 2009,139,2. JFThompson,C.Bellerjeau,G.Marinick,J.Osio-Norgaard,A.Evans,P.Carry,RAStreet,C.Petit,GLWhiting,ACS Applied Materials and Interfaces 2019,11,43337. M. Atreya, K. Dikshit, G. Marinick, J. Nielson, C. Bruns, GL Whiting, ACS Applied Materials and Interfaces 2020,12,23494. D. Eng, M. Caplan, M. Preul, A. Panitch, Acta Biomaterialia 2010,6,2407.

[0042] Definition: As used herein, the term “composition” is intended to encompass any product containing a specific component in a specific amount, and any product resulting directly or indirectly from a specific combination of a specific component in a specific amount.

[0043] "Safe and effective amount" refers to the amount of an ingredient that is sufficient to produce the desired therapeutic response without undesirable adverse side effects (such as toxicity, irritation, or allergic reactions) being proportional to a reasonable benefit / risk ratio when used in the manner of the present invention.

[0044] Throughout this application, the terms “one (a)” and “one (an)” are used to mean “at least one,” “at least one,” “one or more,” or “more than one” of the referenced components or stages, unless the context explicitly specifies otherwise. For example, the term “cell” includes multiple cells, including a mixture thereof.

[0045] Wherever the terms “and / or” are used herein, they include the meanings of “and,” “or,” and “all or any other combination of the elements connected by the terms thereof.”

[0046] As used herein, the terms “about” or “approximately” mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0047] Except in the examples, or unless otherwise clearly specified, the values ​​for the amount of material, reaction time and temperature, quantity ratio, and molecular weight (number average molecular weight ("M") in the following parts of this specification. n ) or weight-average molecular weight ("M w All numerical ranges, quantities, values, and percentages, such as ) ), may be read as if they were prefixed with the word “approximately,” even if the term “approximately” cannot be clearly expressed in a value, quantity, or range. Accordingly, unless otherwise indicated, the numerical parameters described in the subsequent specification and the appended claims are approximations that may vary depending on the desired characteristics sought to be obtained by this disclosure. Where it is precisely the minimum and not an attempt to limit the application of the concept of equivalents to the claims, each numerical parameter shall be interpreted at least in terms of a significant reported value and by applying ordinary rounding techniques.

[0048] Although the numerical ranges and parameters describing the broad scope of this disclosure are approximations, the numerical values ​​described in specific examples are reported as accurately as possible. However, any numerical value inherently contains errors that inevitably result from the standard deviation observed in individual test measurement results of a particular error. Furthermore, when numerical ranges of varying ranges are described herein, it is intended that any combination of values ​​within the numerical range, including the cited values, may be used.

[0049] As used herein, the term “contains” is intended to mean that the product, composition, and method contains the referenced component or step, but not excludes others. “Essentially consisting of” when used to describe the product, composition, and method shall mean excluding any other component or step of essential significance. Thus, a composition essentially consisting of the referenced component would not exclude trace contaminants and pharmaceutically acceptable carriers. “Consists of” shall mean excluding more trace elements than other components or steps.

[0050] Suitable for implantation in patients (humans or animals) means that the biocompatible material is sterilized / finally sterilized, endotoxin-free, and non-cytotoxic. Suitable for use with 3D printers means that the composition provides a smooth flow during extrusion, can be extruded in a pressure range of 20-100 kPa, and polymerizes after extrusion.

[0051] The present invention is not limited in scope by the fact that specific embodiments described, as well as functionally equivalent methods and components intended to describe particular aspects of the invention, fall within the scope of the invention. In fact, various modifications of the invention will be apparent to those skilled in the art from this specification and accompanying drawings, in addition to those shown and described herein. Such modifications are intended to fall within the scope of the claims.

[0052] The advantages described above and those revealed herein so far are efficiently achieved. Since certain modifications can be made in the prior interpretation without departing from the scope of the invention, all matters included herein or shown in the accompanying drawings are intended to be for illustrative purposes only and not to be limited.

[0053] All references cited in this application are incorporated herein by reference to the extent that they are not inconsistent with this specification.

[0054] Since the advantages described above and those revealed herein so far are found to be efficiently achieved, and because certain modifications can be made in the foregoing interpretation without departing from the scope of the invention, all matters included herein or shown in the accompanying drawings are intended to be for illustrative purposes only and not to be limited.

[0055] It is also understood that the subsequent claims are intended to encompass all of the general and specific features of the invention described herein, as well as, as a matter of language, all of the statements that may be said to fall within the scope of the invention. This concludes the description of the invention.

Claims

1. 1. A composition comprising tissue extracellular matrix particles in combination with a thiol-functionalized polymer, the polymer adapted to crosslink to the tissue extracellular matrix particles, thereby forming a biocompatible scaffold.

2. 2. The composition of claim 1, wherein the thiol-functionalized polymer is a non-synthetic natural material selected from the group consisting of collagen, gelatin, hyaluronan, and heparin, polyethylene glycol, or a combination of the thiol-functionalized polymers.

3. 2. The composition of claim 1, wherein the thiol-functionalized polymer has a percentage of functional groups substituted with thiols selected from the group consisting of 0-15%, 15-30%, 30-60%, and combinations thereof.

4. the composition is a tissue mimic; The tissue extracellular matrix particles originate from a native tissue or organ that is a xenogeneic, allogeneic or autologous source to the subject in which the composition is implanted or to which the composition is to be applied; The composition of claim 1.

5. 2. The composition of claim 1, wherein the tissue extracellular matrix particles originate from a tissue selected from the group consisting of liver, kidney, skin, cartilage, bone, muscle, ligament, tendon and fat, and combinations thereof.

6. 2. The composition of claim 1, wherein the tissue extracellular matrix particles are in a size range selected from the group consisting of less than about 100 μm, 100-250 μm, and / or 250-500 μm along their largest dimension.

7. 2. The composition of claim 1, wherein the tissue extracellular matrix particles are decellularized, devitalized and / or lyophilized tissue extracellular matrix particles.

8. 2. The composition of claim 1 , 2. The composition of claim 1, wherein the composition is injected into a tissue space and solidified, such that the solidified composition comprises a lubricious layer consisting essentially of hyaluronan deposited on the surface of the injected composition, whereby the lubricious layer mimics the surface characteristics of natural cartilage.

9. 1. A method of preparing an organoid or tissue mimicking composition, comprising: providing comminuted extracellular matrix particles via mechanical comminution of an original tissue source, thereby obtaining particles that retain the micromechanical and biological properties of said original tissue; combining the extracellular matrix particles with a thiol-functionalized polymer in a hydrogel precursor solution; incubating the hydrogel precursor solution at about 25° C. to about 42° C. for about 10 minutes to about 2 hours to promote disulfide bonds between sulfide groups on the extracellular matrix particles and sulfide groups on the thiol-functionalized polymer, thereby forming an organoid or tissue-mimicking composition; The method includes:

10. The method of reconstructing an organoid or tissue mimicking composition of claim 9, wherein the original tissue source is processed to produce a mimic having living cells or a mimic having decellularized, devitalized, or lyophilized extracellular matrix particles.

11. The method of reconstructing an organoid or tissue-mimicking composition of claim 9, wherein the polymer in the thiol-functionalized polymer has a percentage of functional groups substituted with thiols selected from the group consisting of 0-15%, 15-30%, 30-60%, and combinations thereof.

12. 10. The method of reconstructing organoids or tissue-mimicking compositions of claim 9, wherein the tissue extracellular matrix particles are derived from connective tissue, epithelial tissue or neural tissue and are in a size range selected from the group consisting of less than about 100 μm, 100-250 μm and / or 250-500 μm in the direction of their greatest dimension.

13. The method of reconstructing an organoid or tissue mimicking composition of claim 9, further comprising bioprinting, extruding, layering, or molding the hydrogel solution to form an organoid or tissue mimic having a desired shape or conforming to a void in a subject.

14. The method of reconstructing an organoid or tissue-mimicking composition of claim 9, further comprising the step of implanting or bioprinting the incubated hydrogel into an area of ​​defective, diseased or damaged tissue in a subject.

15. The method of reconstructing an organoid or tissue-mimicking composition of claim 9, wherein the hydrogel precursor solution further comprises one or more of an exogenous growth factor, a region-specific anti-inflammatory drug, or an FDA approved drug.

16. 1. A composition comprising cartilage extracellular matrix particles in combination with a thiol-functionalized hyaluronic acid polymer, the polymer adapted to crosslink to the particles, thereby forming a biocompatible scaffold.

17. 17. The composition of claim 16, wherein the extracellular matrix particles originate from a tissue selected from the group consisting of liver, kidney, skin, bone, muscle, ligament, tendon and fat, and combinations thereof.

18. 17. The composition of claim 16, wherein the thiol-functionalized hyaluronic acid polymer is a non-synthetic natural material selected from the group consisting of collagen, gelatin, and heparin, a synthetic material selected from polyethylene glycol, or a combination thereof.

19. 17. The composition of claim 16, wherein the cartilage extracellular matrix particles are in a size range selected from the group consisting of less than about 100 μm, 100-250 μm, and 250-500 μm along their largest dimension.

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