A bioengineering scaffold and its preparation method, and regenerative tissue

Abstract

The present disclosure provides a bioengineered scaffold, a preparation method thereof, and regenerative tissue, relating to the fields of tissue engineering and regenerative medicine. The bioengineered scaffold provided by the present disclosure retains the complex and unobstructed vascular structure of the target organ. As an engineered material, it has a biomimetic structure and mechanical strength that closely resembles that of a living target organ, supports cell adhesion and growth, facilitates the formation of new functional tissues for medical purposes, and provides new ideas for the construction of in vitro organ function models.

Description

Technical Field

[0001] This specification relates to the technical field of tissue engineering and regenerative medicine, and in particular to a bioengineering scaffold and its preparation method, and regenerative tissue. Background Art

[0002] Liver failure is a serious stage in the progression of various liver diseases, with a high mortality rate and currently no effective treatment. Bioartificial livers play a key role in the treatment of liver failure, but various biomaterials that serve as scaffolds for liver cells lack the unique structure and bioactivity of natural liver tissue. To address this challenge, researchers are exploring bioengineered liver regeneration based on decellularized liver matrix (DLM).

[0003] Decellularization (also known as decellularization) is a method that combines physical and chemical treatments. Decellularization can effectively remove the cellular contents contained in tissues or organs while retaining the components and functional proteins of the extracellular matrix (ECM). Decellularized organs not only have good optical transparency, but also retain tissue-specific three-dimensional ultrastructure and a complete vascular network system. In addition, decellularized organs also retain naturally occurring ECM components, including growth factors, various collagens, and laminin, which have been shown to contribute to the attachment and growth of hepatocytes. For this reason, bioscaffolds prepared from decellularized tissues and organs have been widely used in tissue engineering and regeneration. However, although DLM, as a cell scaffold, retains the liver's unique ultrastructure and various natural extracellular matrix components that are conducive to cell attachment and growth, DLM, which is mainly composed of collagen, lacks effective mechanical support and is extremely prone to collapse, making it difficult to maintain the three-dimensional structure required for hepatocyte culture, making DLM a disadvantage in long-term hepatocyte culture.

[0004] One way to solve the above problem is to mix the HepG2 cell suspension with 5% methacrylate gelatin (GelMA), inject it into DLM, quickly cross-link it with light, and culture it to prepare a bioartificial liver with ammonia conversion function. However, although the method of directly injecting hydrogel into the decellularized liver and cross-linking it improves the mechanical strength of the cell scaffold, the hydrogel blocks the complex vascular structure of the decellularized liver after cross-linking, damages the inherent vascular network of the DLM, thereby reducing the efficiency of nutrient and oxygen delivery during long-term perfusion culture, which is not conducive to the nutrient supply of cells inside the liver. Based on this, it is necessary to provide a bio-engineered scaffold, a preparation method thereof, and regenerative tissue. Summary of the Invention

[0005] This specification provides a bioengineering scaffold and a preparation method thereof, as well as regenerative tissue.

[0006] In a first aspect, the present specification provides a bioengineering scaffold comprising a decellularized tissue matrix and a cross-linked product, wherein the decellularized tissue matrix is obtained by decellularizing at least a portion of a target organ, a tissue matrix space is formed within the decellularized tissue matrix, and the decellularized tissue matrix comprises a vascular wall network matrix and an intervascular network matrix, the vascular wall network matrix forms an intravascular network space, the vascular wall network matrix is obtained by decellularizing the vascular network in at least a portion of the target organ, and the intervascular network matrix is obtained by decellularizing the tissue between the blood vessels in at least a portion of the target organ; the cross-linked product is present in the tissue matrix space excluding the intravascular network space, and the intravascular network space remains unobstructed.

[0007] In some embodiments, the target organ comprises at least one of an internal organ or a sensory organ of the organism.

[0008] In some embodiments, the target organ includes at least one of an animal organ and a human organ.

[0009] In some embodiments, the cross-linked product is a three-dimensional network structure formed by a cross-linked precursor under the initiation of an initiator.

[0010] In some embodiments, the initiator includes a photoinitiator, and the cross-linking precursor undergoes a cross-linking reaction under the initiation of the photoinitiator to form a hydrophilic three-dimensional network structure gel.

[0011] In some embodiments, the cross-linker comprises a biocompatible, degradable cross-linker.

[0012] In some embodiments, the decellularized tissue matrix further comprises: a bile duct wall network matrix, wherein the bile duct wall network matrix forms an intra-bile duct network space, and the bile duct wall network matrix is obtained by decellularizing the bile duct network in at least part of the target organ.

[0013] In a second aspect, the present specification provides a method for preparing a bioengineering scaffold, comprising: obtaining a decellularized tissue matrix; and forming a cross-linked substance in the tissue matrix space other than the intravascular network space, to obtain a bioengineering scaffold in which the intravascular network space remains unobstructed, thereby providing support for the decellularized tissue matrix. In the process of obtaining the decellularized tissue matrix, the decellularized tissue matrix is obtained by decellularizing at least a portion of a target organ, and a tissue matrix space is formed within the decellularized tissue matrix. The decellularized tissue matrix includes a vascular wall network matrix and an intervascular network matrix. The vascular wall network matrix forms an intravascular network space. The vascular wall network matrix is obtained by decellularizing the vascular network in at least a portion of the target organ, and the intervascular network matrix is obtained by decellularizing the tissue between the blood vessels in at least a portion of the target organ.

[0014] In some embodiments, forming a cross-linked product in the tissue matrix space other than the intravascular network space comprises: injecting a solution to be cross-linked into the decellularized tissue matrix space through the intravascular network space, so that the solution to be cross-linked exists in the tissue matrix space other than the intravascular network space, wherein the solution to be cross-linked includes a cross-linking precursor and an initiator; and applying preset reaction conditions to the decellularized tissue matrix to cause the solution to be cross-linked to undergo a curing reaction to form the cross-linked product, wherein the cross-linked product exists in the tissue matrix space other than the intravascular network space.

[0015] In some embodiments, the method of making the solution to be cross-linked exist in the tissue matrix space other than the intravascular network space includes: injecting a vascular filler into the intravascular network space so that the vascular filler replaces the solution to be cross-linked in the intravascular network space, wherein after the cross-linker is formed, the vascular filler is removed from the intravascular network space.

[0016] In some embodiments, the viscosity of the vascular filling material may vary with changes in the external environment.

[0017] In some embodiments, the vascular filler is a thermosensitive hydrogel, which undergoes a reversible change between a solid state and a liquid state as the temperature changes.

[0018] In some embodiments, when the vascular filling material is injected into the intravascular network space, the viscosity of the vascular filling material is higher than the viscosity of the solution to be cross-linked.

[0019] In some embodiments, the preset reaction conditions include reaction conditions that can trigger a photo-crosslinking reaction in the cross-linked solution.

[0020] In some embodiments, the vascular filler is removed from the intravascular network space by injecting water or a buffer solution into the intravascular network space to dilute the vascular filler, and flushing the diluted vascular filler out of the intravascular network space.

[0021] In a third aspect, the present specification provides a regenerative tissue comprising: a bioengineering scaffold and a plurality of target cells, wherein the bioengineering scaffold comprises a decellularized tissue matrix and a crosslinked substance, the decellularized tissue matrix is obtained by decellularizing at least a portion of the target organ, a tissue matrix space is formed in the decellularized tissue matrix, and the decellularized tissue matrix comprises a vascular wall network matrix and an intervascular network matrix, the vascular wall network matrix forms an intravascular network space, the vascular wall network matrix is obtained by decellularizing the vascular network in at least a portion of the target organ, and the intervascular network matrix is obtained by decellularizing the tissue between the blood vessels in at least a portion of the target organ; the crosslinked substance exists in the tissue matrix space other than the intravascular network space, the intravascular network space remains unobstructed, and the plurality of target cells and the crosslinks exist together in the tissue matrix space other than the intravascular network space.

[0022] In some embodiments, the target cells are of a different cell type than the cell type of the at least a portion of the target organ.

[0023] In some embodiments, the target cells are of the same cell type as the cells of the at least a portion of the target organ.

[0024] In some embodiments, the target organ is the liver, the target cells are liver-related cells; and / or the regenerative tissue is a regenerative organ.

[0025] In some embodiments, the liver-related cells include at least one of human hepatoma cells, human cholangiocarcinoma cells, human hepatocytes, human cholangiocarcinoma cells, human hepatic stellate cells, induced pluripotent stem cells, or mesenchymal stem cells.

[0026] In some embodiments, in the regenerated tissue, the decellularized tissue matrix of the bioengineering scaffold further comprises: a bile duct wall network matrix, which forms an intra-bile duct network space, and the bile duct wall network matrix is obtained by decellularizing the bile duct network in at least part of the target organ.

[0027] In some embodiments, the cross-linker comprises a biocompatible, degradable cross-linker.

[0028] In a fourth aspect, the present specification provides a method for preparing regenerative tissue, comprising: obtaining a decellularized tissue matrix, wherein the decellularized tissue matrix is obtained by decellularizing at least a portion of a target organ, a tissue matrix space is formed in the decellularized tissue matrix, and the decellularized tissue matrix includes a vascular wall network matrix and an intervascular network matrix, the vascular wall network matrix forms an intravascular network space, the vascular wall network matrix is obtained by decellularizing the vascular network in at least a portion of the target organ, and the intervascular network matrix is obtained by decellularizing the tissue between the blood vessels in at least a portion of the target organ; thereafter, introducing living cells into the intervascular network matrix; then forming a cross-linked substance in the tissue matrix space other than the intravascular network space to obtain a bioengineering scaffold in which the intravascular network space remains unobstructed, thereby supporting the decellularized tissue matrix, the living cells and the cross-linked substance coexisting in the tissue matrix space other than the intravascular network space; finally, culturing the living cells on the bioengineering scaffold to obtain a regenerative tissue containing multiple target cells.

[0029] In some embodiments, the target cells are of a different cell type than the cell type of the at least a portion of the target organ.

[0030] In some embodiments, the target cells are of the same cell type as the cells of the at least a portion of the target organ.

[0031] In some embodiments, the introducing living cells into the intervascular network matrix comprises: injecting a suspension containing the living cells into the acellular tissue matrix space through the intravascular network space, and the living cells enter the intervascular network matrix through the vascular wall network matrix.

[0032] In some embodiments, the decellularized tissue matrix of the bioengineering scaffold further comprises: a bile duct wall network matrix, which forms an intra-bile duct network space, and the bile duct wall network matrix is obtained by decellularizing the bile duct network in at least a portion of the target organ; at this time, the step of introducing living cells into the intervascular network matrix comprises: injecting a suspension containing the living cells into the decellularized tissue matrix space through the intra-bile duct network space, and the living cells enter the intervascular network matrix through the bile duct wall network matrix.

[0033] In some embodiments, forming a cross-linked product in the tissue matrix space other than the intravascular network space comprises: injecting a solution to be cross-linked into the decellularized tissue matrix space through the intravascular network space, so that the solution to be cross-linked exists in the tissue matrix space other than the intravascular network space, wherein the solution to be cross-linked includes a cross-linking precursor and an initiator; and applying preset reaction conditions to the decellularized tissue matrix to cause the solution to be cross-linked to undergo a curing reaction to form the cross-linked product, wherein the cross-linked product exists in the tissue matrix space other than the intravascular network space.

[0034] In some embodiments, the method of making the solution to be cross-linked exist in the tissue matrix space other than the intravascular network space includes: injecting a vascular filler into the intravascular network space so that the vascular filler replaces the solution to be cross-linked in the intravascular network space, wherein after the cross-linker is formed, the vascular filler is removed from the intravascular network space.

[0035] In some embodiments, the vascular filler is removed from the intravascular network space by injecting cell culture fluid into the intravascular network space to dilute the vascular filler, and flushing the diluted vascular filler out of the intravascular network space.

[0036] It can be seen from the above technical solutions that in the bioengineering scaffold provided in this specification, the cross-linked material exists in the tissue matrix space other than the intravascular network space, which retains the complex and unobstructed vascular structure contained in the target organ, so that nutrients can circulate smoothly in the intravascular network space. In addition, the cross-linked material has better mechanical strength than the decellularized tissue matrix, and can support the decellularized tissue matrix to prevent the decellularized tissue matrix from collapsing. Therefore, the bioengineering scaffold provided in the present disclosure, as an engineered material, has a biomimetic structure and mechanical strength that is highly similar to the target organ in a living body, and can support cell adhesion and growth, which helps to form new functional tissues for medical purposes, and can provide new ideas for the construction of in vitro organ function models. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of this specification, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of this specification. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0038] Figure 1 is a flow chart of a method for preparing a bioengineering scaffold according to some embodiments of the present specification;

[0039] Figure 2 A flow chart of a method for preparing regenerated tissue according to some embodiments of the present specification;

[0040] Figure 3A A diagram of a fresh liver state used in some embodiments of the present specification;

[0041] Figure 3B is a diagram of the state of a fresh liver after processing according to some embodiments of the present specification;

[0042] Figure 3C is a state diagram of a decellularized liver according to some embodiments of the present specification;

[0043] Figure 4 is a schematic diagram of a bioreactor according to some embodiments of the present specification;

[0044] Figure 5 A flow chart for preparing a regenerated liver according to some embodiments of the present specification;

[0045] Figure 6 is a diagram of the liver state after ink perfusion according to some embodiments of the present specification;

[0046] Figure 7 is another diagram of a liver state after ink perfusion according to some embodiments of the present specification;

[0047] Figure 8 This is a diagram of a liver internal tissue section according to some embodiments of the present specification;

[0048] Figure 9 is another diagram of an internal tissue section of the liver according to some embodiments of the present specification;

[0049] Figure 10 Mechanical strength test results of fresh liver (FL), decellularized liver (DLM) and regenerated liver (GLM) according to some embodiments of the present specification; and

[0050] Figure 11 These are the water retention test results of fresh liver (FL), decellularized liver (DLM) and regenerated liver (GLM) according to some embodiments of the present specification. DETAILED DESCRIPTION

[0051] To facilitate understanding of this specification, a more comprehensive description of the specification will be provided below with reference to the accompanying drawings. The drawings illustrate preferred embodiments of this specification. However, this specification can be implemented in many different forms without departing from the core spirit of this specification and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of this specification.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this specification pertains. The terms used herein in this specification are for the purpose of describing specific embodiments only and are not intended to limit this specification. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0053] For the convenience of description, the terms that may appear in this manual are first explained as follows:

[0054] Cross-linking reaction: refers to the reaction in which two or more molecules (usually linear molecules) bond and cross-link to form a relatively stable molecule (bulk molecule) network structure. This reaction transforms linear or slightly branched macromolecules into a three-dimensional network structure.

[0055] Cross-linked products: also known as cross-linked polymers and cross-linked macromolecules, are a type of polymer with a three-dimensional network structure.

[0056] Hydrogels are a type of extremely hydrophilic gel with a three-dimensional network structure. They swell rapidly in water and, in this swollen state, can retain large volumes of water without dissolving. Any water-soluble or hydrophilic polymer can form a hydrogel through certain chemical or physical crosslinking. Due to the crosslinked network, hydrogels can swell and retain large amounts of water, and the amount of water absorbed is closely related to the degree of crosslinking. The higher the degree of crosslinking, the lower the water absorption. This characteristic is very similar to that of soft tissue. The water content in a hydrogel can range from a few percent to as high as 99%. The aggregated state of a gel is neither completely solid nor completely liquid. Solid behavior is the ability to maintain a specific shape and volume under certain conditions, while liquid behavior is the ability for solutes to diffuse or permeate through the hydrogel.

[0057] In a first aspect, this specification provides a bioengineering scaffold comprising a decellularized tissue matrix and a cross-linked material, wherein the decellularized tissue matrix is obtained by decellularizing at least a portion of a target organ, forming tissue matrix spaces within the decellularized tissue matrix, and further comprising a vascular wall network matrix and an intervascular network matrix. The vascular wall network matrix is obtained by decellularizing the vascular network in at least a portion of the target organ, forming intravascular network spaces within the vascular wall network matrix; and the intervascular network matrix is obtained by decellularizing tissue between blood vessels in at least a portion of the target organ.

[0058] The cross-linked substance exists in the tissue matrix space except the intravascular network space, and the intravascular network space remains unobstructed.

[0059] The decellularized tissue matrix disclosed in the present invention is obtained by decellularizing the target organ. The decellularized tissue matrix comprises the complete vascular network system of the target organ (equivalent to the vascular wall network matrix) and the extracellular matrix components other than the vascular network system (equivalent to the intervascular network matrix). Since there are some gaps inside the target organ itself (such as gaps between cells or other gaps), and after the target organ is decellularized, the original cells are removed, and more gaps are formed in the decellularized tissue matrix. These gaps form the tissue matrix space of the decellularized tissue matrix, and thus the decellularized tissue matrix presents a loose three-dimensional structure. In the decellularized tissue matrix, the above-mentioned vascular wall network matrix forms an intravascular network space.

[0060] In the bioengineered scaffolds provided herein, crosslinks are present in the tissue matrix space, in addition to the intravascular network space. Therefore, the bioengineered scaffolds retain the complex and unobstructed vascular structure of the target organ, allowing nutrients to circulate freely within the intravascular network space. Furthermore, because the crosslinks possess greater mechanical strength than the decellularized tissue matrix, they can support the decellularized tissue matrix and prevent its collapse.

[0061] It should be noted that the cross-linked material disclosed herein possesses a three-dimensional network structure due to its inherent physicochemical properties. Furthermore, due to its unique location (existing within the tissue matrix space outside the intravascular network), the cross-linked material exhibits a similar loose three-dimensional structure to the loose structure of the acellular tissue matrix within this space.

[0062] In the present disclosure, there is also a gap between the cross-linked material and the decellularized tissue matrix. The size of the gap between the cross-linked material and the decellularized tissue matrix can be controlled by regulating the amount of the cross-linked material. Generally speaking, the more cross-linked materials that exist in the tissue matrix space other than the intravascular network space, the less space they occupy, and the smaller the gap between the cross-linked material and the decellularized tissue matrix; conversely, the fewer cross-linked materials that exist in the tissue matrix space other than the intravascular network space, the more space they occupy, and the larger the gap between the cross-linked material and the decellularized tissue matrix. The gaps between the decellularized tissue matrices, the gaps in the cross-linked materials themselves, and the gaps between the cross-linked materials and the decellularized tissue matrix can all accommodate cells and provide space for cell growth.

[0063] The bioengineered scaffold provided in the present disclosure is an engineered material with a biomimetic structure and mechanical strength that is highly similar to a living target organ, and can support cell adhesion and growth, thereby helping to form new functional tissues for medical purposes and providing new ideas for the construction of in vitro organ function models.

[0064] The bioengineered scaffolds provided herein can be used alone, for example, as an in vitro organ model to aid in pharmacokinetics research in the context of sustained drug release. The bioengineered scaffolds provided herein can also be used in combination with cells, for example, as a cell culture scaffold to form regenerative biological tissues.

[0065] For ease of understanding, the space formed by the intervascular network matrix can be referred to as the extravascular network space, and the intravascular network space and the extravascular network space together constitute the tissue matrix space of the acellular tissue matrix. In this case, the tissue matrix space excluding the intravascular network space is the extravascular network space.

[0066] In the present disclosure, the target organ can be a structure with a certain morphology and function formed by several different types of tissues that have developed, differentiated and combined with each other. The target organ can be classified according to different standards. At the same time, in order to simplify the description, the organs or partial organ tissues are collectively referred to as "organs" in the present disclosure. That is to say, the "organ" in the present disclosure can refer to the organ itself, or to a partial organ and / or organ tissue. When the context allows, the technology involved allows, and / or the creativity allows, the "organ" should be understood in a broader sense.

[0067] In some embodiments, the target organ may be classified according to its functional type, and may include all or part of at least one of an organism's internal organs or sensory organs. Internal organs may include the heart, liver, lungs, stomach, kidneys, etc.; sensory organs may include the eyes, ears, nose, tongue, etc.; and target organs may also include all or part of skeletal muscle, skin, and other organs with specific morphology and function.

[0068] In some embodiments, the target organ can be classified according to its source, and can include at least one of an animal organ and a human organ. Animal organs can be obtained from experimental animals (e.g., mice, rats, ferrets, rabbits, dogs, pigs, monkeys, etc.); human organs can be obtained from legitimate sources (e.g., heart, liver, spleen, lungs, kidneys, etc.).

[0069] In some embodiments, the target organs may be classified according to the system category of the organs. For example, the target organ may be all or part of an organ included in at least one of the following systems: the skeletal system, the muscular system, the digestive system, the ligamentous system, the respiratory system, the urinary system (such as the kidneys), the circulatory system (such as the heart), and the lymphatic system (such as the spleen).

[0070] It should be understood that the above examples of target organ types do not limit the actual target organ types disclosed herein. Any organ that can be decellularized and has a vascular network structure after decellularization is within the scope of the target organ to be protected by the present disclosure.

[0071] It should be understood that the amount of crosslinker used in the bioengineering scaffolds provided herein can be considered in conjunction with factors such as the source of the decellularized tissue matrix and the size of the decellularized tissue matrix itself. For example, decellularized tissue matrices derived from different target organs may have different sizes, vascular network structures, and the amount of crosslinker used may vary accordingly. Those skilled in the art can adjust the amount of crosslinker used according to actual conditions to adapt to decellularized tissue matrices from different sources.

[0072] In the present disclosure, a crosslinking precursor can be used to undergo a crosslinking reaction under the action of an initiator, solidifying to form a three-dimensional network structure, thereby introducing a crosslinked product into tissue matrix spaces other than the intravascular network space. Both the crosslinking precursor and the initiator can enter the tissue matrix space in the form of a fluid. When the loading amounts of the crosslinking precursor and the initiator are within a certain range, suitable reaction conditions can be applied so that the crosslinking precursor, under the initiator's action, crosslinks and solidifies to form a three-dimensional network structure with a certain mechanical strength, i.e., a crosslinked product.

[0073] In some embodiments, the initiator comprises a photoinitiator, and the crosslinking precursor undergoes a crosslinking reaction under the initiation of the photoinitiator to form a hydrophilic three-dimensional network structure gel. For example, the hydrophilic three-dimensional network structure gel can be any hydrogel that supports photocrosslinking. Hydrogels can swell in water and retain a large amount of water without dissolving, thereby providing the bioengineering scaffold with good water retention properties.

[0074] The hydrophilic three-dimensional network structure gel (also called hydrogel) can include the following five categories according to the type of intermediate:

[0075] (1) Free radical hydrogels. This type of hydrophilic three-dimensional network structure gel can receive light energy under light through a photoinitiator, change from the ground state to the excited state, and then decompose into free radicals. The free radicals combine with the carbon-carbon double bond of the cross-linking precursor, and on this basis, chain growth occurs, causing the carbon-carbon double bond to polymerize. Among them, the photoinitiator can include phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO), 2,4,6-trimethylbenzoylphenyl phosphonic acid ethyl ester (TPO-L), 2-methyl-1-[4-methylthiophenyl]-2-morpholinyl-1-propanone (907), 2-isopropylthioxanthone (ITX), 1-hydroxycyclohexyl phenyl ketone (184), 2-hydroxy-2-methylpropiophenone (1173), 2,2- Dimethoxy-2-phenylacetophenone (BDK), methyl 2-benzoylbenzoate (OMBB), benzophenone (BP), 4-chlorobenzophenone (CBP), 4-phenylbenzophenone (PBZ), 2-phenylbenzyl-2-dimethylamino-1-(4-morphophenylphenyl)butanone (369), 1,1'-(methylenebis-4,1-phenylene)bis[2-hydroxy-2-methyl-1-propanone] (127), ethyl 4-dimethylaminobenzoate (EDB), isooctyl p-dimethylaminobenzoate (EHA) ), 4-methylbenzophenone (MBZ), 2,4-diethylthioxanthene-9-one (DETX), 2-(4-methylbenzyl)-2-(dimethylamino)-1-(4-morpholinophenyl)-1-butanone (379), 4-benzoyl-4'-methyl-diphenyl sulfide (BMS), 2,2'-bis(o-chlorophenyl)-4,4',5,5'-tetraphenyl-1,2'-bisimidazole (BCIM), 2,2'-bis(o-chlorophenyl)-4,4',5,5'-tetraphenyl-1,2'-bisimidazole At least one of oxazole (OXE01), 1-[4-(4-benzoylphenylthio)phenyl]-2-toluenesulfonyl-2-methyl-1-propanone (1001M), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (2959), methyl benzoylformate (MBF), or 2-ethylanthraquinone (EAQ); the cross-linking precursor may include a gel with double bond modification, such as acrylic anhydride-modified gel, methacrylic anhydride-modified gel, and / or methacrylic acid glycidaldehyde-modified gelatin, etc.

[0076] (2) Cationic polymer hydrogels: This type of hydrophilic three-dimensional network structure gel can use the proton acid generated by cationic photoinitiators under light to catalyze the ring-opening polymerization of epoxy groups or the cationic polymerization of electron-rich carbon-carbon double bonds. Among them, the photoinitiator can include triphenylsulfonium hexafluorophosphate, diphenyliodonium hexafluorophosphate (810), 4-phenylthiophenyl diphenylsulfonium salt, tris[4-[(4-acetylphenyl)thio]phenyl]-hexafluorophosphate (Irgacure 209), 4-dodecyloxyphenyl diphenylsulfonium hexafluoroantimonate (SOC 10), 9-[4-(2-hydroxyethoxy)-phenyl]thianthrenesulfonium hexafluorophosphate (Esacure 1187), 10-(4-biphenyl)-2-isopropylthioxanthonesulfonium hexafluorophosphate (Omnicat 550), [7-(1-methylethyl)-9-oxo-9H-thioxanthen-2yl]bis(4-methylphenyl)sulfonium hexafluoroantimonate (PCI 061T), bis[(4-diphenylsulfonium)phenyl]sulfide-bis-hexafluorophosphate (UV 6992), 4-phenylthiophenyl diphenylsulfonium salt, 4,4-bis(thiazolin-9-yl)biphenyl hexafluorophosphate, S,S'-(thiodi-4,1-phenylene)bis[S,S-bis[4-(2-hydroxyethoxy)phenyl]sulfonium hexafluorophosphate (antimonate) (SP150), (4-hydroxyphenyl)methyl(benzyl)hexasulfonium fluorophosphate (PHS SI100L), 4-acetoxyphenyldimethylsulfonium hexafluoroantimonate (Sanaid SI 150), (4-hydroxyphenyl)methyl[(2-methylphenyl)methyl]-sulfonium hexafluoroantimonate (Sanaid 80L), dodecylmethyl(2-oxo-2-phenylethyl)-sulfonium hexafluoroantimonate, diphenyliodonium hexafluorophosphate (Photoinitiator 810), bis(4-methylbenzene)iodonium hexafluorophosphate, bis(4-dodecylbenzene)iodonium hexafluorophosphate, bis(4-isopropylbenzene)iodonium hexafluorophosphate, bis(4-tert-butylbenzene)iodonium hexafluorophosphate, (4-methylphenyl)[4-(2-methylpropyl)phenyliodonium hexafluorophosphate (Irgacure 250), or 4-octyloxyphenyliodonium hexafluoroantimonate (UVACURE 1600); the cross-linking precursor may include at least one of gelatin modified with groups such as epoxy resin, vinyl ether, alicyclic epoxy, and oxetane, chondroitin sulfate, agarose, hyaluronic acid, chitosan, or sodium alginate.

[0077] (3) Nitrile hydrogels: The cross-linking precursors that can be used for this type of hydrogel include chitosan modified with azide groups, chondroitin sulfate, sodium alginate, gelatin, agarose, and / or hyaluronic acid.

[0078] (IV) Carbene hydrogels: Cross-linking precursors that can be used for this type of hydrogel include gelatin modified with diazo groups or diazirine groups, sodium alginate, chondroitin sulfate, chitosan, agarose, and / or hyaluronic acid.

[0079] (5) Carbon cation hydrogels. The cross-linking precursors that can be used for this type include gelatin modified with aromatic diazonium salts, sodium alginate, chondroitin sulfate, chitosan, agarose, and / or hyaluronic acid.

[0080] In some embodiments, the crosslinker comprises a biocompatible, degradable crosslinker. The degradable crosslinker can decompose in a biological environment and has low cytotoxicity. When living cells are implanted on the bioengineered scaffold of the present disclosure, the crosslinker gradually decreases in volume during degradation, providing more space for the living cells to grow, promoting their growth, proliferation, and differentiation, thereby providing favorable conditions for their growth.

[0081] In addition to high water permeability and water retention, hydrogels are also bioadhesive, biodegradable, and biocompatible. Furthermore, hydrogels possess mechanical strength similar to that of soft tissue in living organisms. Therefore, the crosslinked materials disclosed herein can be made of hydrogels.

[0082] In some embodiments, the cross-linking precursor can be methacrylated gelatin (GelMA). Methacrylated gelatin is a photosensitive biomaterial that can be rapidly cross-linked and cured under blue light or ultraviolet light when used in conjunction with a photoinitiator to form a three-dimensional network structure hydrogel with a certain strength. The hydrogel has good water retention properties; the hydrogel structure has cell adhesion sites and matrix metalloproteinase hydrolysis sites, which can well support cell proliferation and migration, and can load a variety of cells such as tumor cells, cardiomyocytes, and chondrocytes; and the mechanical properties of the cross-linked hydrogel can be adjusted by changing the degree of substitution and concentration of methacrylated gelatin, thereby improving its mechanical structure and thus improving the mechanical properties of the bioengineering scaffold.

[0083] In some embodiments, the decellularized tissue matrix further comprises a bile duct wall network matrix, which forms intra-bile duct network spaces, and the bile duct wall network matrix is obtained by decellularizing the bile duct network of at least a portion of the target organ. When the target organ is the liver, since the liver also includes bile ducts, the decellularized tissue matrix obtained by decellularizing the liver also includes the bile duct network system of the liver (equivalent to the bile duct wall network matrix).

[0084] In a second aspect, this specification also provides a method for preparing a bioengineering scaffold, such as Figure 1 As shown, the preparation method comprises the following steps:

[0085] S110, obtaining a decellularized tissue matrix. The decellularized tissue matrix can be obtained by decellularizing at least a portion of the target organ. Tissue matrix spaces are formed within the decellularized tissue matrix, and the decellularized tissue matrix includes a vascular wall network matrix and an intervascular network matrix. The vascular wall network matrix is obtained by decellularizing the vascular network in at least a portion of the target organ. The vascular wall network matrix forms intravascular network spaces; the intervascular network matrix is obtained by decellularizing tissue between blood vessels in at least a portion of the target organ.

[0086] As mentioned above, the decellularized tissue matrix contains the complete vascular network system of the target organ (equivalent to the vascular wall network matrix) and the extracellular matrix components other than the vascular network system (equivalent to the intervascular network matrix). Since there are some gaps inside the target organ itself (such as gaps between cells or other gaps), and after the target organ is decellularized, the original cells are removed, and more gaps are formed in the decellularized tissue matrix. These gaps form the tissue matrix space of the decellularized tissue matrix. In the decellularized tissue matrix, the above-mentioned vascular wall network matrix forms the intravascular network space.

[0087] S120, forming a cross-linked substance in the tissue matrix space except the intravascular network space, to obtain a bioengineering scaffold in which the intravascular network space remains unobstructed, so as to support the decellularized tissue matrix.

[0088] In the present disclosure, after obtaining a decellularized tissue matrix, a cross-linked material can be formed at specific locations within the decellularized tissue matrix (within the tissue matrix space other than the intravascular network space), thereby obtaining a bioengineered scaffold that maintains an unobstructed intravascular network space, allowing nutrients to circulate freely within the intravascular network space. Furthermore, because the cross-linked material has greater mechanical strength than the decellularized tissue matrix, it can support the decellularized tissue matrix and prevent its collapse.

[0089] Therefore, by using the preparation method of the bioengineering scaffold provided in the present disclosure, a bioengineering scaffold with a biomimetic structure and mechanical strength that is highly similar to the target organ in living body can be prepared, thereby helping to form new functional tissues for medical purposes and providing new ideas for the construction of in vitro organ function models.

[0090] In some embodiments, the method of forming a cross-linked substance in the tissue matrix space other than the intravascular network space may be to use a cross-linking precursor to generate a cross-linked substance having a three-dimensional network structure under the initiation of an initiator, which may specifically include:

[0091] Injecting a solution to be cross-linked into the decellularized tissue matrix space through the intravascular network space, and allowing the solution to be cross-linked to exist in the tissue matrix space excluding the intravascular network space, wherein the solution to be cross-linked includes a cross-linking precursor and an initiator; and

[0092] Preset reaction conditions are applied to the decellularized tissue matrix to cause the cross-linked solution to undergo a solidification reaction to form the cross-linked product, which exists in the tissue matrix space except the intravascular network space.

[0093] In the present disclosure, the cross-linking precursor includes a polymer compound that has not been cross-linked and cured, and the cross-linking precursor can undergo a cross-linking and curing reaction under the initiation of an initiator to form a polymer (cross-linked product) with a three-dimensional network structure. The cross-linking precursor and the initiator can be dissolved in a specific solvent to form a solution to be cross-linked. Since the intravascular network space in the decellularized tissue matrix is an internal space formed by the vascular wall network matrix, the vascular wall network matrix is obtained by decellularization of the complete vascular network system in the target organ. The blood vessels themselves have a certain permeability, which allows liquids, gases and macromolecular substances to pass through selectively, and the blood vessels also have other types of pore structures. Therefore, whether it is a vascular wall that has not been decellularized or a vascular wall network matrix obtained after decellularization, it has a pore structure, which allows substances with a diameter smaller than the pore structure to pass through the vascular wall or the vascular wall network matrix. Moreover, the vascular network system of the target organ includes various blood vessels, such as arteries, veins, capillaries, etc. When the cross-linking solution is injected into the decellularized tissue matrix space through the intravascular network space, the cross-linking precursor and initiator can flow along the direction of the vascular network, pass through the vascular wall network matrix, enter, and be evenly distributed in the internal space of the decellularized tissue matrix.

[0094] Since the tissue matrix space formed by the decellularized tissue matrix includes the intravascular network space and the tissue matrix space outside the intravascular network space, the location of the crosslinked product formed after crosslinking and curing can be controlled by controlling the location of the crosslinked solution in the tissue matrix space during the crosslinking reaction. In the present disclosure, the solution to be crosslinked is present in the tissue matrix space outside the intravascular network space. At this time, the solution to be crosslinked does not exist in the intravascular network space. When the preset reaction conditions are applied to the decellularized tissue matrix, the crosslinked product formed by the crosslinking and curing reaction of the solution to be crosslinked is present in the tissue matrix space outside the intravascular network space.

[0095] The preset reaction conditions mentioned above include reaction conditions that can cause the crosslinking precursor and the initiator to undergo a crosslinking and curing reaction. For example, the preset reaction conditions include reaction conditions that can induce the solution to be crosslinked to undergo a photocrosslinking reaction.

[0096] In some embodiments, the method of making the solution to be cross-linked exist in the tissue matrix space other than the intravascular network space includes: injecting a vascular filler into the intravascular network space so that the vascular filler replaces the solution to be cross-linked in the intravascular network space, wherein after the cross-linker is formed, the vascular filler is removed from the intravascular network space.

[0097] After the solution to be cross-linked is injected into the decellularized tissue matrix space through the intravascular network space, the cross-linking precursor and initiator flow along the direction of the vascular network, pass through the vascular wall network matrix, enter, and are evenly distributed in the internal space of the decellularized tissue matrix. This means that the solution to be cross-linked can exist in the intravascular network space, or in the tissue matrix space other than the intravascular network space. If the solution to be cross-linked exists in the intravascular network space, the cross-linked product formed after the solution to be cross-linked undergoes cross-linking and solidification will exist in the intravascular network space. At this time, the cross-linked product will occupy the space in the intravascular network and cause blockage. If there is too much solution to be cross-linked in the intravascular network space, the cross-linked product formed after the solution to be cross-linked undergoes cross-linking and solidification may also damage the vascular wall network matrix due to volume expansion.

[0098] In the present disclosure, in order to make the solution to be cross-linked exist only in the tissue matrix space other than the intravascular network space as much as possible, vascular fillers can be continuously injected into the intravascular network space after the solution to be cross-linked is injected. Under the squeezing of the later injected vascular fillers, the previously injected solution to be cross-linked continuously diffuses through the vascular wall network matrix into the tissue matrix space other than the intravascular network space. When the amount of vascular fillers injected later reaches a certain amount, the solution to be cross-linked previously existing in the intravascular network space can be replaced. At this time, there is basically only vascular fillers in the intravascular network space, and the solution to be cross-linked is removed from the intravascular network space and squeezed into the tissue matrix space other than the intravascular network space. The vascular filler is not sensitive to the reaction conditions that trigger the cross-linking and curing of the solution to be cross-linked. Therefore, after the solution to be cross-linked is cross-linked and cured to form a cross-linked material, the vascular filler is removed from the intravascular network space to form a bioengineering scaffold in which the intravascular network space remains unobstructed.

[0099] In some embodiments, when the vascular filling material is injected into the intravascular network space, the viscosity of the vascular filling material is higher than the viscosity of the solution to be cross-linked.

[0100] Viscosity, also known as viscosity, refers to a fluid's resistance to flow. It characterizes the consistency of a substance; the harder a substance flows, the higher its viscosity. When viscosity exceeds a certain level, the substance becomes solid.

[0101] The vascular filler and the solution to be cross-linked described in this disclosure are both injected into the intravascular network space. The difference lies in the order of injection: the solution to be cross-linked is injected into the intravascular network space before the vascular filler. In this disclosure, to ensure that the solution to be cross-linked is smoothly squeezed into the tissue matrix space outside the intravascular network space, the viscosity of the vascular filler and the solution to be cross-linked is controlled so that the viscosity of the vascular filler is higher than that of the solution to be cross-linked. In this case, the fluidity of the solution to be cross-linked is superior to that of the vascular filler, making it easier for the solution to cross-linked to penetrate the interstices of the vascular wall network matrix. Furthermore, as previously mentioned, the solution to be cross-linked is injected into the intravascular network space before the vascular filler. Since the later-injected vascular filler has a higher viscosity, it continuously occupies the volume previously occupied by the solution to be cross-linked in the intravascular network space during the continuous injection process. This effectively applies pressure to the solution to be cross-linked, causing it to continuously penetrate the interstices of the vascular wall network matrix. The intravascular network space is ultimately occupied by the vascular filler, and the solution to be cross-linked is present in the tissue matrix space outside the intravascular network space.

[0102] It should be noted that, since the present disclosure squeezes the cross-linked solution into the tissue matrix space other than the intravascular network space by injecting a vascular filler into the intravascular network space, during this process, a very small amount of vascular filler may also pass through the gaps in the vascular wall network matrix and enter the tissue matrix space other than the intravascular network space.

[0103] In some embodiments, the vascular filler is removed from the intravascular network space by injecting water or a buffer solution into the intravascular network space to dilute the vascular filler, and then flushing the diluted vascular filler out of the intravascular network space.

[0104] In the present disclosure, after the cross-linked solution in the tissue matrix space outside the intravascular network space has completed cross-linking and solidification to form a cross-linked material with a three-dimensional network structure, the originally high-viscosity vascular filler can be diluted to reduce its viscosity, for example, by continuously injecting water or a buffer solution into the intravascular network space. After the viscosity of the vascular filler is reduced, its fluidity is enhanced, and it can now pass through the gaps in the network matrix of the vascular wall. The newly injected water or buffer solution also creates pressure for the vascular filler to diffuse outward. The vascular filler continues to diffuse outward with the water flow and is eventually removed from the intravascular network space. At this point, the substance present in the intravascular network space is replaced by water or a buffer solution.

[0105] It should be understood that, after the vascular filler has been removed from the intravascular network, if water or buffer solution is continuously injected into the intravascular network, the vascular filler present in the tissue matrix space outside the intravascular network will be continuously squeezed out of its original space under the action of external pressure, continuing to flow outward with the water flow. As a result, the vascular filler present in the tissue matrix space outside the intravascular network will also be continuously diluted or even removed.

[0106] In some embodiments, the viscosity of the vascular filling material may vary with changes in the external environment.

[0107] In the present disclosure, a substance whose viscosity changes with the external environment is selected as a vascular filler, and the temperature of the vascular filler can be controlled to achieve the desired viscosity of the vascular filler. For example, when injecting the vascular filler into the intravascular network space, the vascular filler can be controlled to maintain a low viscosity state within a certain temperature range so that it can be smoothly injected into the intravascular network space. After the vascular filler is injected into the intravascular network space, the temperature of the vascular filler can be adjusted to increase its viscosity. Accordingly, the fluidity is reduced. At this time, the vascular filler is less likely to pass through the vascular wall network matrix than the solution to be cross-linked already existing in the intravascular network space. Therefore, the vascular filler with increased viscosity can promote the solution to be cross-linked to continuously pass through the vascular wall network matrix and enter the tissue matrix space outside the intravascular network space, while the vascular filler itself remains in the intravascular network space due to its poor fluidity.

[0108] In some embodiments, the vascular filler can be an injectable thermosensitive hydrogel that can undergo a reversible change between a solid state and a liquid state with changes in temperature. For example, the thermosensitive hydrogel can include sodium alginate, hyaluronic acid, sodium alginate modified with methylcellulose or hydroxypropyl methylcellulose, hyaluronic acid; sodium alginate, cellulose, hyaluronic acid modified with chitosan; chitosan, thioglycolic acid conjugate (CS-TGA); chitosan-4-mercaptobutyramidine conjugate (CS-TBA); PEO-PPO-PEO triblock copolymer (trade name P407); PLGA-PEG copolymer; N-isopropylacrylamide PNIPAAm modified products, including PNIPAAm modified acrylamide (AAm), N,N-dimethylacrylamide (DMAAm), methacrylamide (MAAm), chiral monomer N-(L-1-ethoxycarbonyl-2-phenyl) acrylamide (AAc-l-pheEt), hydrophobic monomer N-(N-tert-butyloxycarbonyl-ethylenediamine) glycine dipeptide methacrylamide (BRMAGG), 2-(dimethylamino)ethyl-methacrylamide (2-DMAEMAAm), acrylic acid (AAc), methacrylic acid (MAA), sodium methacrylate (SMAA), (3-sulfonic acid)propyl potassium salt of acrylic acid (SPAPS), (3-sulfonic acid)propyl potassium salt of methacrylate (SPMAPS), hydroxypropyl methacrylate (HPMA), methyl acrylate (MA), ethyl acrylate (EA), butyl acrylate (BA), tert-butyl acrylate (tBA), β-hydroxyethyl methacrylate (HEMA), starch maleate (starch-MAH), dextran grafted polycaprolactone-hydroxyethyl methacrylate (Dex-PCL-HEMA), N-vinyl pyrrolidone (NVP), polyethylene glycol monomethacrylate (PEGMA), sodium carboxymethylcellulose (CMC), alginate (sodium, calcium), guar gum (GG), sericin, chitosan (CS), konjac glucomannan (KGM), or at least one of a dextran derivative (GMA-Dex).

[0109] As previously mentioned, the bioengineered scaffolds provided herein can be used alone or in combination with cells. The following description will use the bioengineered scaffolds provided herein in combination with cells as an example. It should be understood that the use of the bioengineered scaffolds provided herein in combination with cells does not limit other uses of the bioengineered scaffolds provided herein.

[0110] In a third aspect, this specification also provides a regenerative tissue, comprising a bioengineering scaffold and a plurality of target cells. The bioengineering scaffold comprises a decellularized tissue matrix and a crosslinked product. The decellularized tissue matrix is obtained by decellularizing at least a portion of the target organ, and a tissue matrix space is formed within the decellularized tissue matrix. Furthermore, the decellularized tissue matrix comprises a vascular wall network matrix and an intervascular network matrix. The vascular wall network matrix is obtained by decellularizing the vascular network in at least a portion of the target organ, and the vascular wall network matrix forms an intravascular network space; the intervascular network matrix is obtained by decellularizing the tissue between the blood vessels in at least a portion of the target organ. The crosslinked product is present in the tissue matrix space excluding the intravascular network space, and the intravascular network space remains unobstructed. The plurality of target cells and the crosslinked product are present together in the tissue matrix space excluding the intravascular network space.

[0111] It is understandable that the regenerated tissue may be a whole regenerated organ. For example, when the acellular tissue matrix is obtained by decellularizing the whole target organ, the regenerated tissue is a whole regenerated organ.

[0112] In the present disclosure, the bioengineering scaffold that constitutes the regenerative tissue can be understood as a cell culture scaffold. Since in the bioengineering scaffold, the cross-linked material exists in the tissue matrix space other than the intravascular network space, the complex and unobstructed vascular structure contained in the target organ is retained, so that nutrients can circulate freely in the intravascular network space. In addition, the cross-linked material has better mechanical strength than the decellularized tissue matrix, and can support the decellularized tissue matrix to prevent the decellularized tissue matrix from collapsing. The above-mentioned multiple target cells and the cross-linked material coexist in the tissue matrix space other than the intravascular network space, and the multiple target cells surround the cross-linked material. It can also be understood that the multiple target cells surround the decellularized tissue matrix. The cross-linked material provides support for the decellularized tissue matrix while also providing support for the multiple target cells.

[0113] In some cases, the plurality of target cells can be understood as the cells contained in the target organ before decellularization. Therefore, the bioengineered scaffold and the plurality of target cells together constitute the regenerated tissue described in the present disclosure, which can serve as an in vitro model of the original target organ, exhibiting the same morphology and performing the same function as the target organ.

[0114] In the present disclosure, as mentioned above, target organs can be classified according to different standards.

[0115] When categorized by organ function, the target organ can include all or part of at least one of a living organism's internal organs or sensory organs. Internal organs can include the heart, liver, lungs, stomach, and kidneys, while sensory organs can include the eyes, ears, nose, and tongue. Furthermore, the target organ can also include all or part of an organ with a specific morphology and function, such as skeletal muscle or skin.

[0116] When categorized by organ source, the target organs can include all or part of animal organs and / or human organs. Animal organs can be sourced from experimental animals (e.g., mice, rats, ferrets, rabbits, dogs, pigs, monkeys, etc.); human organs can be sourced from legitimate sources (e.g., heart, liver, spleen, lungs, kidneys, etc.).

[0117] In addition, the target organs can also be divided according to the system category of the organs. For example, the target organ can be all or part of an organ included in at least one of the following systems: the skeletal system, the muscular system, the digestive system, the ligament system, the respiratory system, the urinary system (such as the kidneys), the circulatory system (such as the heart), and the lymphatic system (such as the spleen).

[0118] It should be understood that the above examples of target organ types do not limit the actual target organ types disclosed herein. Any organ that can be decellularized and has a vascular network structure after decellularization is within the scope of the target organ to be protected by the present disclosure.

[0119] In some embodiments, in the regenerated tissue, the decellularized tissue matrix of the bioengineering scaffold may further include a bile duct wall network matrix, which forms a bile duct intra-network space, and the bile duct wall network matrix is obtained by decellularizing the bile duct network in at least a portion of the target organ. As previously mentioned, when the target organ is the liver, since the liver also includes bile ducts, the decellularized tissue matrix obtained by decellularizing the liver organ also includes the bile duct network system of the liver organ (equivalent to the bile duct wall network matrix). Furthermore, the regenerated tissue cultured using the bioengineering scaffold may also include the bile duct network system (equivalent to the bile duct wall network matrix).

[0120] Because the bioengineered scaffolds included in the regenerative tissues provided herein retain the complex and unobstructed vascular structure of the organ, nutrients can circulate freely within the intravascular network. Therefore, under appropriate external nutrient supply conditions, various target cell types can attach to the bioengineered scaffolds and carry out normal physiological activities, achieving growth and differentiation. In the present disclosure, the target cells may be of the same or different cell types as the target organ.

[0121] In some embodiments, the cell type of the target cell is different from the cell type of at least part of the target organ. In the present disclosure, when the cell type of the target cell is different from the cell type of the target organ, other types of cells can be cultured using a bioengineering scaffold to obtain various in vitro models with scientific value. For example, if the bioengineering scaffold is obtained by decellularization of the liver, the target cells may be other types of cells other than liver-related cells, such as blood vessel-related cells (such as vascular endothelial cells), brain-related cells (such as neural stem cells), soft tissue-related cells (such as muscle stem cells, adipose stem cells, induced pluripotent stem cells, etc.), bone tissue-related cells (such as osteoblasts, osteoclasts, mesenchymal stem cells), etc. If the bioengineering scaffold is obtained by decellularization of the heart, the target cells may be other types of cells other than heart-related cells.

[0122] It should be understood that the bioengineering scaffold can also be obtained from other organs through decellularization, and the target cells can also be other types of cells, which are not listed here one by one.

[0123] In some embodiments, the target cells are of the same cell type as the cells of the at least a portion of the target organ.

[0124] As mentioned above, the multiple target cells can be understood as the cells contained in the target organ before decellularization. Therefore, the cell types of the multiple target cells can be the same as the cell types contained in the target organ. For example, if the target organ is the liver, the cell types of the target cells can be the same as the cell types contained in the liver. In this case, the target cells can be referred to as liver-related cells, and the regenerated tissue is correspondingly regenerated liver tissue. The liver-related cells can include at least one of human hepatoma cells, human cholangiocarcinoma cells, human hepatocytes, human cholangiocarcinoma cells, human hepatic stellate cells, induced pluripotent hepatocytes, mesenchymal stem cells, and the like.

[0125] If the target organ is the heart, the cell type of the target cells can be the same as the cell type contained in the heart. In this case, the target cells can be called heart-related cells, and correspondingly, the regenerated tissue is regenerated heart tissue; if the target organ is other organs, the cell type of the target cells can be the same as the cell type contained in the corresponding organ.

[0126] In the present disclosure, the target cells may be cells obtained by growing, proliferating and differentiating other types of cells on the bioengineering scaffold. The target cells may also be attached to the bioengineering scaffold and carry out normal cell physiological activities, such as growth, proliferation and differentiation, with the support of the unobstructed vascular structure of the bioengineering scaffold. The attachment of the target cells to the bioengineering scaffold can be understood as a plurality of target cells being attached to the cross-linked material with a three-dimensional network structure contained in the bioengineering scaffold, or as target cells being distributed in the gaps in the tissue matrix space other than the intravascular network space. The bioengineering scaffold not only provides space for the target cells to carry out normal physiological activities, but also allows the target cells to be distributed in various positions, avoiding the target cells from agglomerating, so that the formed regenerated tissue is close to the original target organ in terms of morphology and function.

[0127] In some embodiments, the crosslinker comprises a biocompatible, degradable crosslinker. When the target cells attach to the bioengineering scaffold and, supported by the unobstructed vascular structure of the bioengineering scaffold, carry out normal cellular physiological activities, the degradable crosslinker can be decomposed. Furthermore, the degradable crosslinker exhibits excellent biocompatibility and does not harm the target cells in the regenerated tissue. During degradation, the crosslinker's volume gradually decreases, providing more growth space for the target cells and promoting the growth, proliferation, and differentiation of living cells.

[0128] In the present disclosure, the biocompatible, degradable cross-linked material can be selected from hydrogels. In addition to having high water permeability and water retention, hydrogels also exhibit bioadhesiveness, biodegradability, and good biocompatibility. Furthermore, hydrogels possess mechanical strength similar to that of biological soft tissue. Depending on the type of intermediate, the hydrogel can be selected from at least one of free radical hydrogels, cationic polymer hydrogels, nitrene hydrogels, carbene hydrogels, and carbon cation hydrogels.

[0129] Among them, hydrogels using methacrylated gelatin (GelMA) as a cross-linking precursor are photosensitive biomaterials. When used with a photoinitiator, GelMA can rapidly crosslink and cure under blue or ultraviolet light, forming a hydrogel with a strong three-dimensional network structure. This hydrogel has excellent water retention properties and contains cell adhesion sites and matrix metalloproteinase hydrolysis sites within its structure, which can effectively support cell proliferation and migration and can load a variety of cells such as tumor cells, cardiomyocytes, and chondrocytes. Furthermore, the mechanical properties of the cross-linked hydrogel can be adjusted by varying the degree of substitution and concentration of GelMA, improving its mechanical structure and, consequently, the mechanical properties of bioengineering scaffolds.

[0130] In a fourth aspect, this specification also provides a method for preparing regenerated tissue, such as Figure 2 As shown, the preparation method includes:

[0131] S210, obtaining a decellularized tissue matrix, wherein the decellularized tissue matrix is obtained by decellularizing the target organ, wherein tissue matrix spaces are formed within the decellularized tissue matrix, and wherein the decellularized tissue matrix comprises a vascular wall network matrix and an intervascular network matrix. The vascular wall network matrix is obtained by decellularizing the vascular network in the target organ, wherein the vascular wall network matrix forms intravascular network spaces; and the intervascular network matrix is obtained by decellularizing tissue between blood vessels in the target organ.

[0132] In the present disclosure, the decellularized tissue matrix of the target organ is obtained by decellularizing the target organ. As mentioned above, the decellularized tissue matrix contains the complete vascular network system of the target organ (equivalent to the vascular wall network matrix) and the extracellular matrix components other than the vascular network system (equivalent to the intervascular network matrix). Since there are some gaps inside the target organ itself (such as gaps between cells or other gaps), and after the target organ is decellularized, the original cells are removed, and there are many gaps in the formed decellularized tissue matrix. These gaps form the tissue matrix space of the decellularized tissue matrix. In the decellularized tissue matrix, the above-mentioned vascular wall network matrix forms the intravascular network space.

[0133] S220, introducing living cells into the intervascular network matrix;

[0134] As previously mentioned, since the intravascular network space in the decellularized tissue matrix is the internal space formed by the vascular wall network matrix, the vascular wall network matrix is obtained by decellularizing the complete vascular network system in the target organ. Since the blood vessels themselves have a certain permeability, they can selectively allow liquids, gases, and macromolecules to pass through, and the blood vessels also have other types of pore structures. Therefore, whether it is a vascular wall that has not been decellularized or a vascular wall network matrix obtained after decellularization, it has a pore structure, which allows substances with a diameter smaller than the pore structure to pass through the vascular wall or the vascular wall network matrix. The diameter of living cells is generally in the micron level, which can pass through the pores in the vascular wall or the vascular wall network matrix.

[0135] Therefore, in some embodiments, the introducing living cells into the intervascular network matrix may include: injecting a suspension containing the living cells into the acellular tissue matrix space through the intravascular network space, and the living cells enter the intervascular network matrix through the vascular wall network matrix.

[0136] S230, forming a cross-linked substance in the tissue matrix space excluding the intravascular network space, to obtain a bioengineering scaffold in which the intravascular network space remains unobstructed, thereby supporting the decellularized tissue matrix, wherein the living cells and the cross-linked substance coexist in the tissue matrix space excluding the intravascular network space;

[0137] In the present disclosure, after introducing living cells into the intervascular network matrix, a cross-linked material is formed at a specific location in the decellularized tissue matrix (within the tissue matrix space other than the intravascular network space), thereby obtaining a bioengineering scaffold in which the intravascular network space remains unobstructed. Because the cross-linked material has better mechanical strength than the decellularized tissue matrix, it can support the decellularized tissue matrix and prevent the decellularized tissue matrix from collapsing. Furthermore, the intravascular network space of the bioengineering scaffold remains unobstructed, allowing nutrients to circulate freely in the intravascular network space, thereby providing life support for the living cells present in the tissue matrix space other than the intravascular network space.

[0138] In the present disclosure, considering that the size of living cells is much larger than that of substances such as cross-linking precursors, initiators, and vascular fillers, the living cells are injected into the intervascular network matrix of the decellularized tissue matrix before the above substances are injected into the decellularized tissue matrix. This not only ensures that the living cells can smoothly enter the intervascular network matrix through the vascular wall matrix and form a uniform distribution, thereby successfully culturing regenerative tissue with substantially the same structure and function as the target organ; but also, injecting the living cells first will not significantly affect the activity of subsequent substances (such as cross-linking precursors, initiators, vascular fillers, etc.) in the decellularized tissue matrix.

[0139] Because cells easily diffuse from the bile duct into the liver parenchyma and grow densely, in some embodiments, when the regenerated tissue is a liver organ, introducing living cells into the intervascular network matrix may include: injecting a suspension containing the living cells into the acellular tissue matrix space through the intra-biliary network space, whereby the living cells enter the intervascular network matrix through the bile duct wall network matrix.

[0140] In some embodiments, the method of forming a cross-linked substance in the tissue matrix space other than the intravascular network space may be to use a cross-linking precursor to generate a cross-linked substance having a three-dimensional network structure under the initiation of an initiator, which may specifically include:

[0141] Injecting a solution to be cross-linked into the decellularized tissue matrix space through the intravascular network space, so that the solution to be cross-linked exists in the tissue matrix space except the intravascular network space, wherein the solution to be cross-linked includes a cross-linking precursor and an initiator;

[0142] Preset reaction conditions are applied to the decellularized tissue matrix to cause the cross-linked solution to undergo a solidification reaction to form the cross-linked product, which exists in the tissue matrix space except the intravascular network space.

[0143] In the present disclosure, the cross-linking precursor includes a polymer compound that has not been cross-linked and cured, and the cross-linking precursor can undergo a cross-linking and curing reaction under the initiation of an initiator to form a polymer (cross-linked product) with a three-dimensional network structure. The cross-linking precursor and the initiator can be dissolved in a solvent to form a solution to be cross-linked. Since the intravascular network space in the decellularized tissue matrix is an internal space formed by the vascular wall network matrix, the vascular wall network matrix is obtained by decellularization of the complete vascular network system in the target organ. Since the blood vessels themselves have a certain permeability, liquids, gases and macromolecular substances can be selectively passed through, and the blood vessels also have other types of pore structures. Therefore, whether it is a vascular wall that has not been decellularized or a vascular wall network matrix obtained after decellularization, it has a pore structure, which can allow substances with a diameter smaller than the pore structure to pass through the vascular wall or the vascular wall network matrix. Moreover, the vascular network system of the target organ includes various blood vessels, such as arteries, veins, capillaries, etc. When the cross-linking solution is injected into the decellularized tissue matrix space through the intravascular network space, the cross-linking precursor and initiator can flow along the direction of the vascular network, pass through the vascular wall network matrix, enter, and be evenly distributed in the internal space of the decellularized tissue matrix.

[0144] Since the tissue matrix space formed by the decellularized tissue matrix includes the intravascular network space and the tissue matrix space excluding the intravascular network space, the location of the cross-linked product formed after cross-linking and curing can be controlled by controlling the location of the cross-linked solution in the tissue matrix space when the cross-linking reaction occurs. In the present disclosure, the solution to be cross-linked is made to exist in the tissue matrix space outside the intravascular network space. At this time, the solution to be cross-linked does not exist in the intravascular network space. When the preset reaction conditions are applied to the decellularized tissue matrix, the solution to be cross-linked undergoes a cross-linking and curing reaction, and the cross-linked product formed exists in the tissue matrix space excluding the intravascular network space.

[0145] The preset reaction conditions mentioned above include reaction conditions that can cause the crosslinking precursor and the initiator to undergo a crosslinking and curing reaction. For example, the preset reaction conditions include reaction conditions that can induce the solution to be crosslinked to undergo a photocrosslinking reaction.

[0146] In some embodiments, the method of making the solution to be cross-linked exist in the tissue matrix space other than the intravascular network space may include: injecting a vascular filler into the intravascular network space so that the vascular filler replaces the solution to be cross-linked in the intravascular network space, wherein, after the cross-linker is formed, the vascular filler is removed from the intravascular network space.

[0147] After the solution to be cross-linked is injected into the decellularized tissue matrix space through the intravascular network space, the cross-linking precursor and initiator flow along the direction of the vascular network, pass through the vascular wall network matrix, enter, and are evenly distributed in the internal space of the decellularized tissue matrix. This means that the solution to be cross-linked can exist in the intravascular network space or in the tissue matrix space other than the intravascular network space. If the solution to be cross-linked exists in the intravascular network space, the cross-linked product formed after the cross-linked solution undergoes cross-linking and solidification will exist in the intravascular network space. At this time, the cross-linked product will occupy the space in the intravascular network and cause blockage. If the intravascular network space is filled with the solution to be cross-linked, the cross-linked product formed after the cross-linked solution undergoes cross-linking and solidification may also damage the vascular wall network matrix due to volume expansion.

[0148] In the present disclosure, in order to ensure that the solution to be cross-linked exists only in the tissue matrix space other than the intravascular network space as much as possible, a vascular filler can be continuously injected into the intravascular network space after the solution to be cross-linked is injected. Under the pressure of the later injected vascular filler, the previously injected solution to be cross-linked continuously diffuses through the vascular wall network matrix into the tissue matrix space other than the intravascular network space. When the later injected vascular filler reaches a certain amount, it can replace the solution to be cross-linked previously existing in the intravascular network space. At this time, the intravascular network space basically only contains the vascular filler, and the solution to be cross-linked is removed from the intravascular network space and squeezed into the tissue matrix space other than the intravascular network space. The vascular filler is insensitive to the reaction conditions that trigger the cross-linking and curing of the solution to be cross-linked. Therefore, after the solution to be cross-linked is cross-linked and cured to form a cross-linked material, the vascular filler is removed from the intravascular network space to form a bioengineering scaffold with an unobstructed intravascular network space.

[0149] In some embodiments, when the vascular filling material is injected into the intravascular network space, the viscosity of the vascular filling material is higher than the viscosity of the solution to be cross-linked.

[0150] Viscosity, also known as viscosity, refers to a fluid's resistance to flow. It characterizes the consistency of a substance; the harder a substance flows, the higher its viscosity. When viscosity exceeds a certain level, the substance becomes solid.

[0151] The vascular filler and the solution to be cross-linked described in the present disclosure are both injected into the intravascular network space, differing in the order of injection: the solution to be cross-linked is injected into the intravascular network space before the vascular filler. In the present disclosure, to ensure smooth extrusion of the solution to be cross-linked into the tissue matrix space outside the intravascular network space, the viscosities of the vascular filler and the solution to be cross-linked are controlled so that the viscosity of the vascular filler is higher than that of the solution to be cross-linked. In this case, the fluidity of the solution to be cross-linked is superior to that of the vascular filler, allowing the solution to more easily penetrate the interstices of the vascular wall network matrix. Furthermore, as previously described, the solution to be cross-linked is injected into the intravascular network space before the vascular filler. Due to the greater viscosity of the later-injected vascular filler, it continuously displaces the volume previously occupied by the solution to be cross-linked in the intravascular network space during the injection process, effectively applying pressure to the solution to be cross-linked, causing it to continuously penetrate the interstices of the vascular wall network matrix. Ultimately, the intravascular network space is occupied by the vascular filler, leaving the solution to be cross-linked in the tissue matrix space outside the intravascular network space.

[0152] It should be noted that, since the present disclosure squeezes the cross-linked solution into the tissue matrix space other than the intravascular network space by injecting a vascular filler into the intravascular network space, during this process, a very small amount of vascular filler may also pass through the gaps in the vascular wall network matrix and enter the tissue matrix space other than the intravascular network space.

[0153] In some embodiments, the vascular filler is removed from the intravascular network space by injecting cell culture fluid into the intravascular network space to dilute the vascular filler, and flushing the diluted vascular filler out of the intravascular network space.

[0154] In the present disclosure, after the cross-linked solution in the tissue matrix space outside the intravascular network space has completed cross-linking and solidified to form a cross-linked product with a three-dimensional network structure, the originally highly viscous vascular filler can be diluted. Because living cells have already been introduced into the decellularized tissue matrix before the vascular filler is injected into the intravascular network space during the preparation of regenerated tissue, to ensure that the vascular filler does not affect the living cells during removal from the intravascular network space, cell culture fluid can be continuously injected into the intravascular network space to dilute the vascular filler and reduce its viscosity. With the reduced viscosity of the vascular filler, its fluidity increases, allowing it to pass through the gaps in the vascular wall network matrix. The newly injected cell culture fluid also exerts pressure on the vascular filler to diffuse outward. The vascular filler continues to diffuse outward with the flow of the cell culture fluid and is ultimately removed from the intravascular network space. At this point, the substance present in the intravascular network space is replaced by the cell culture fluid.

[0155] It should be understood that, after the vascular filler has been removed from the intravascular network, if cell culture fluid is continuously injected into the intravascular network, the vascular filler present in the tissue matrix space outside the intravascular network will be continuously squeezed out of its original space under the action of external pressure, and will continue to flow outward with the cell culture fluid. As a result, the vascular filler present in the tissue matrix space outside the intravascular network space will also be continuously diluted or even removed.

[0156] S240, culturing the living cells on the bioengineering scaffold to obtain regenerative tissue containing a plurality of target cells.

[0157] In the present disclosure, multiple target cells can be derived from living cells that have been previously injected into a decellularized tissue matrix and grown, proliferated, and differentiated. Furthermore, the multiple target cells can continue to adhere to the bioengineered scaffold and, supported by the bioengineered scaffold's unobstructed vascular structure, carry out normal cellular physiological activities, such as growth, proliferation, and differentiation.

[0158] In the present disclosure, the target cells may be understood as various tissue cells that can attach to the bioengineering scaffold and perform normal physiological activities. Therefore, the cell types of the target cells may be the same as or different from the cell types of the target organ.

[0159] In some embodiments, the target cells are of a different type than the target organ's cells. In this case, a bioengineered scaffold can be used to culture various cell types to produce various models of scientific value.

[0160] In some embodiments, the target cells may be of the same cell type as the target organ. In this case, the target cells may be understood to be cells present in the target organ before decellularization, so that the regenerated tissue can serve as an in vitro model of the original target organ, exhibiting the same morphology and function as the target organ.

[0161] The following are specific preparation examples involved in the above contents of this disclosure. It should be clear that the following examples are only for the purpose of illustrating the bioengineering scaffold, the method for preparing the bioengineering scaffold, the regenerative tissue and the method for preparing the regenerative tissue disclosed above, and the specific implementation methods and parameters used therein are only one or a concentrated method among the many processes and methods described above. Those skilled in the art can use other parameters according to the contents introduced in this specification to prepare the bioengineering scaffold and the regenerative tissue according to the above method without deviating from the core spirit disclosed in the application. For example, the following examples use the liver as the target organ. Those skilled in the art can fully understand that the above-mentioned bioengineering scaffold, the method for preparing the bioengineering scaffold, the regenerative tissue and the method for preparing the regenerative tissue can also be applied to other organs, such as heart, skin, bone, kidney, lung tissue, etc. It is only due to space limitations that the present disclosure does not provide a detailed description of the embodiments of other organs.

[0162] Example 1

[0163] This embodiment provides a method for preparing a bioengineering scaffold, comprising the following steps:

[0164] (1) Preparation of decellularized liver: SD rats weighing 400-500 g were taken and euthanized by inhalation of carbon dioxide. The abdominal cavity was opened along the midline of the rat's abdomen, and the renal vein was tied with cotton thread. A needle was inserted into the bile duct, portal vein, and hepatic vein and tied with cotton thread. The above-mentioned vessels were cut, and the mucosa connecting the liver (FL) and the other organs on the inner wall was separated. The liver was placed in a culture dish for preservation and frozen at -20°C for 24 hours. The liver was removed and thawed at room temperature. The portal vein of the liver was connected to a peristaltic pump and flushed with water, 4% Triton X-100 + 0.02% EGTA solution, and 1% SDS solution at a flow rate of 4 mL / min for 30 minutes, 1 hour, and 3 hours, respectively, until the liver became transparent and the vascular structure was visible, thereby obtaining a decellularized liver (DLM). Figure 3A The state diagram of fresh liver (FL) of SD rat is shown. Figure 3B The figure shows the state of the liver after washing with 4% Triton X-100 + 0.02% EGTA solution. Figure 3C shows the state of the liver after washing with 1% SDS solution, i.e., the state of the decellularized liver (DLM);

[0165] (2) Prepare the cross-linking solution (GelMA solution) and the vascular filler (sodium alginate) solution: weigh 0.3 g of GelMA lyophilized powder, 2.7 mL of 1×PBS, and 300 μL of photoinitiator (2.5% w / v solution), mix well, and incubate in a 65°C water bath until completely dissolved to obtain the GelMA solution. Pass the solution through a 22 μm bacteria filter while hot and set aside; weigh 0.06 g of sodium alginate and 3 mL of distilled water, mix well to prepare a 0.02% w / v sodium alginate aqueous solution, incubate in a 65°C water bath until completely dissolved, sterilize at high temperature, and set aside;

[0166] (3) Preparation of bioengineering scaffolds: 1×PBS was perfused and cleaned through the portal vein of the decellularized liver for 4 h. After cleaning, approximately 5 mL of DEME was injected into the decellularized liver to ensure that the decellularized liver was completely filled with DMEM. The decellularized liver was placed in a 37°C cell culture incubator and incubated for 30 min. 37°C preheated GelMA solution was injected through the portal vein until the entire decellularized liver was filled. 37°C preheated Alginate solution was quickly injected into the portal vein and hepatic vein. The entire liver was cross-linked under ultraviolet light for 30 s. Physiological saline or 1×PBS was injected into the portal vein and hepatic vein to rinse and remove sodium alginate, thereby obtaining a gelled bioengineering scaffold that retained a smooth vascular structure.

[0167] Example 2

[0168] This embodiment provides a preparation method for regenerating a liver, comprising the following steps:

[0169] (1) Preparation of decellularized liver: SD rats weighing 400-500 g were taken and euthanized by inhalation of carbon dioxide. The abdominal cavity was opened along the midline of the rat's abdomen, and the renal vein was tied with cotton thread. A needle was inserted into the bile duct, portal vein, and hepatic vein and tied with cotton thread. The above-mentioned vessels were cut, and the mucosa connecting the liver (FL) and the other organs on the inner wall was separated. The liver was placed in a culture dish for preservation and frozen at -20°C for 24 hours. The liver was removed and thawed at room temperature. The portal vein of the liver was connected to a peristaltic pump and flushed with water, 4% Triton X-100 + 0.02% EGTA solution, and 1% SDS solution at a flow rate of 4 mL / min for 30 minutes, 1 hour, and 3 hours, respectively, until the liver became transparent and the vascular structure was visible, thereby obtaining a decellularized liver (DLM). Figure 3A The state diagram of fresh liver (FL) of SD rat is shown. Figure 3B The figure shows the state of the liver after washing with 4% Triton X-100 + 0.02% EGTA solution. Figure 3C shows the state of the liver after washing with 1% SDS solution, i.e., the state of the decellularized liver (DLM);

[0170] (2) Prepare the cross-linking solution (GelMA solution) and the vascular filler (sodium alginate) solution: weigh 0.3 g of GelMA lyophilized powder, 2.7 mL of 1×PBS, and 300 μL of photoinitiator (2.5% w / v solution), mix well, and incubate in a 65°C water bath until completely dissolved to obtain the GelMA solution. Pass the solution through a 22 μm bacteria filter while hot and set aside; weigh 0.06 g of sodium alginate and 3 mL of distilled water, mix well to prepare a 0.02% w / v sodium alginate aqueous solution, incubate in a 65°C water bath until completely dissolved, sterilize at high temperature, and set aside;

[0171] (3) Preparation of regenerated liver (GLM): 1×PBS was perfused into the portal vein of the decellularized liver for 4 h. After washing, approximately 5 mL of DEME was injected into the decellularized liver to ensure that the decellularized liver was completely filled with DMEM. The decellularized liver was placed in a 37°C cell culture incubator and incubated for 30 min. 4 mL of HepG2 cell suspension was injected into the bile duct of the decellularized liver. The total number of cells was approximately 1×10 8 Then, inject 37℃ preheated GelMA solution from the portal vein until the entire decellularized liver is filled, quickly inject 37℃ preheated Alginate solution into the portal vein and hepatic vein, crosslink the whole liver under ultraviolet light for 30s, inject cell culture medium suitable for HepG2 cells into the portal vein and hepatic vein, rinse to remove sodium alginate, let it stand for 1 hour, wait for cells to attach, and place the liver in Figure 4 After 48 h of culture in the bioreactor shown, a gelled regenerated liver with a clear vascular structure was obtained.

[0172] Figure 5 The preparation process of the regenerated liver in Example 2 is shown.

[0173] Figure 6 The liver is shown in Figure 1, where the lower lobular vascular network is not preserved, and the liver is shown in Figure 2, where ink is perfused into the portal vein and hepatic vein. Figure 7 The diagram shows the state of the regenerated liver prepared in Example 2 after ink was perfused into the portal vein and hepatic vein. Figure 8 This is a slice of liver tissue showing the inner part of the liver without preserving the vascular network of the lower lobule. Figure 8 The scale bar is 100 μm; Figure 9 The figure shows the internal tissue section of the regenerated liver prepared in Example 2. Figure 9 The scale bar is 100 μm. Figure 6 and Figure 7 It can be seen that Figure 7 The ink in the liver can continue to diffuse along the liver's vascular network, and Figure 7 A clear liver vascular network structure (including capillaries) can be seen in Figure 6 The ink dispersion in the middle and lower leaflets is uneven, and the ink dispersion in other parts is slightly higher, but the dispersion is much lower than Figure 7 The dispersion of ink. Figure 8 and Figure 9 It can be seen that Figure 8 The blood vessels are blocked by the gel. Figure 9 The blood vessels are unobstructed.

[0174] Figure 10 The results of mechanical strength tests on fresh liver (FL), decellularized liver (DLM) and regenerated liver (GLM) in Example 2 are shown. Figure 10 It can be seen that the Young's modulus of the regenerated liver (GLM) prepared in Example 2 is slightly higher than that of the fresh liver (FL).

[0175] Figure 11 The results of the water retention test on fresh liver (FL), decellularized liver (DLM) and regenerated liver (GLM) in Example 2 are shown. Figure 11 It can be seen that the regenerated liver (GLM) prepared in Example 2 has a water retention capacity that is substantially the same as that of the fresh liver (FL).

[0176] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above-mentioned embodiments only express several embodiments of the present invention. The description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A bioengineering scaffold, characterized in that: include: The decellularized tissue matrix is obtained by decellularizing at least a portion of the target organ, wherein a tissue matrix space is formed in the decellularized tissue matrix, and the decellularized tissue matrix comprises: A vascular wall network matrix is formed with an intravascular network space, wherein the vascular wall network matrix is obtained by decellularizing the vascular network in at least part of the target organ, and an intervascular network matrix obtained by decellularizing intervascular tissue in at least a portion of the target organ; and The cross-linked substance exists in the tissue matrix space except the intravascular network space, and the intravascular network space remains unobstructed.

2. The bioengineering scaffold according to claim 1, wherein The target organ includes at least one of an internal organ or a sensory organ of a living body.

3. The bioengineering scaffold according to claim 1, wherein: The target organ includes at least one of an animal organ and a human organ.

4. The bioengineering scaffold according to claim 1, wherein: The cross-linked product is a three-dimensional network structure formed by a cross-linked precursor under the initiation of an initiator.

5. The bioengineering scaffold according to claim 4, characterized in that: The initiator includes a photoinitiator, and the cross-linking precursor undergoes a cross-linking reaction under the initiation of the photoinitiator to form a hydrophilic three-dimensional network structure gel.

6. The bioengineering scaffold according to claim 1, wherein: The cross-linked material includes a biocompatible and degradable cross-linked material.

7. The bioengineering scaffold according to claim 1, wherein: The decellularized tissue matrix further comprises: The bile duct wall network matrix forms a bile duct network space, and the bile duct wall network matrix is obtained by decellularizing the bile duct network in at least part of the target organ.

8. A method for preparing a bioengineering scaffold, characterized in that: include: Obtain a decellularized tissue matrix, wherein the decellularized tissue matrix is obtained by decellularizing at least a portion of the target organ, wherein a tissue matrix space is formed within the decellularized tissue matrix, and the decellularized tissue matrix comprises: A vascular wall network matrix is formed with an intravascular network space, wherein the vascular wall network matrix is obtained by decellularizing the vascular network in at least part of the target organ, and an intervascular network matrix obtained by decellularizing intervascular tissue in at least a portion of the target organ; and A cross-linked substance is formed in the tissue matrix space except the intravascular network space to obtain a bioengineering scaffold in which the intravascular network space remains unobstructed, so as to support the decellularized tissue matrix.

9. The preparation method according to claim 8, wherein The forming of cross-links in the tissue matrix space excluding the intravascular network space comprises: Injecting a solution to be cross-linked into the decellularized tissue matrix space through the intravascular network space, and allowing the solution to be cross-linked to exist in the tissue matrix space excluding the intravascular network space, wherein the solution to be cross-linked includes a cross-linking precursor and an initiator; and Preset reaction conditions are applied to the decellularized tissue matrix to cause the cross-linked solution to undergo a solidification reaction to form the cross-linked product, which exists in the tissue matrix space except the intravascular network space.

10. The preparation method according to claim 9, wherein The method of making the cross-linking solution exist in the tissue matrix space other than the intravascular network space includes: injecting a vascular filler into the intravascular network space so that the vascular filler replaces the solution to be cross-linked in the intravascular network space, Wherein, after the cross-linked material is formed, the vascular filler is removed from the intravascular network space.

11. The preparation method according to claim 10, characterized in that The viscosity of the vascular filling material may change with changes in the external environment.

12. The preparation method according to claim 10 or 11, characterized in that: The vascular filler is a thermosensitive hydrogel, which undergoes a reversible change between a solid state and a liquid state as the temperature changes.

13. The preparation method according to claim 10, wherein When the vascular filling material is injected into the intravascular network space, the viscosity of the vascular filling material is higher than the viscosity of the solution to be cross-linked.

14. The preparation method according to claim 10, wherein The preset reaction conditions include reaction conditions that can trigger a photo-crosslinking reaction in the solution to be cross-linked.

15. The preparation method according to claim 10, wherein The method of removing the vascular filler from the intravascular network space includes: Water or a buffer solution is injected into the intravascular network space to dilute the vascular filling material, and the diluted vascular filling material is flushed out of the intravascular network space.

16. A regenerated tissue, characterized in that: include: Bioengineered scaffolds, including: The decellularized tissue matrix is obtained by decellularizing at least a portion of the target organ, wherein a tissue matrix space is formed in the decellularized tissue matrix, and the decellularized tissue matrix comprises: A vascular wall network matrix is formed with an intravascular network space, wherein the vascular wall network matrix is obtained by decellularizing the vascular network in at least part of the target organ, and an intervascular network matrix obtained by decellularizing tissue between blood vessels in at least a portion of the target organ, and Cross-linked substances are present in the tissue matrix space except the intravascular network space, and the intravascular network space remains unobstructed; and A plurality of target cells exist together with the cross-linked substance in the tissue matrix space excluding the intravascular network space.

17. The regenerated tissue according to claim 16, wherein The cell type of the target cells is different from the cell type of the at least part of the target organ.

18. The regenerated tissue according to claim 16, wherein The target cells are of the same type as the cells of at least a portion of the target organ.

19. The regenerated tissue according to claim 18, wherein The target organ is the liver, and the target cells are liver-related cells; and / or The regenerated tissue is a regenerated organ.

20. The regenerated tissue according to claim 19, wherein The liver-related cells include at least one of human hepatoma cells, human cholangiocarcinoma cells, human liver cells, human cholangiocarcinoma cells, human hepatic stellate cells, induced pluripotent hepatocytes or mesenchymal stem cells.

21. The regenerated tissue according to claim 19, wherein The decellularized tissue matrix of the bioengineering scaffold further comprises: The bile duct wall network matrix forms a bile duct network space, and the bile duct wall network matrix is obtained by decellularizing the bile duct network in at least part of the target organ.

22. The regenerated tissue according to claim 16, wherein The cross-linked material includes a biocompatible and degradable cross-linked material.

23. A method for preparing regenerated tissue, characterized in that: include: Obtain a decellularized tissue matrix, wherein the decellularized tissue matrix is obtained by decellularizing at least a portion of the target organ, wherein a tissue matrix space is formed within the decellularized tissue matrix, and the decellularized tissue matrix comprises: A vascular wall network matrix is formed with an intravascular network space, wherein the vascular wall network matrix is obtained by decellularizing the vascular network in at least part of the target organ, and An intervascular network matrix is obtained by decellularizing intervascular tissue in at least a portion of the target organ; introducing living cells into the intervascular network matrix; forming a cross-linked substance in the tissue matrix space excluding the intravascular network space to obtain a bioengineering scaffold in which the intravascular network space remains unobstructed, thereby supporting the decellularized tissue matrix, wherein the living cells and the cross-linked substance coexist in the tissue matrix space excluding the intravascular network space; and The living cells are cultured on the bioengineering scaffold to obtain regenerative tissue containing a plurality of target cells.

24. The preparation method according to claim 23, wherein The cell type of the target cells is different from the cell type of the at least part of the target organ.

25. The preparation method according to claim 23, wherein The target cells are of the same type as the cells of at least a portion of the target organ.

26. The preparation method according to claim 23, wherein The method of introducing living cells into the intervascular network matrix comprises: A suspension containing the living cells is injected into the acellular tissue matrix space through the intravascular network space, and the living cells pass through the vascular wall network matrix and enter the intervascular network matrix.

27. The preparation method according to claim 23, wherein The decellularized tissue matrix of the bioengineering scaffold further comprises: a bile duct wall network matrix, which forms a bile duct intra-network space, and the bile duct wall network matrix is obtained by decellularizing the bile duct network in at least a portion of the target organ; and The method of introducing living cells into the intervascular network matrix comprises: A suspension containing the living cells is injected into the acellular tissue matrix space through the intra-biliary network space, and the living cells pass through the bile duct wall network matrix and enter the inter-vascular network matrix.

28. The preparation method according to claim 23, wherein The forming of cross-links in the tissue matrix space excluding the intravascular network space comprises: Injecting a solution to be cross-linked into the decellularized tissue matrix space through the intravascular network space, so that the solution to be cross-linked exists in the tissue matrix space excluding the intravascular network space, wherein the solution to be cross-linked includes a cross-linking precursor and an initiator; and Preset reaction conditions are applied to the decellularized tissue matrix to cause the cross-linked solution to undergo a solidification reaction to form the cross-linked product, which exists in the tissue matrix space except the intravascular network space.

29. The preparation method according to claim 28, wherein The method of making the cross-linking solution exist in the tissue matrix space other than the intravascular network space includes: injecting a vascular filler into the intravascular network space so that the vascular filler replaces the solution to be cross-linked in the intravascular network space, Wherein, after the cross-linked material is formed, the vascular filler is removed from the intravascular network space.

30. The preparation method according to claim 29, wherein The method of removing the vascular filler from the intravascular network space includes: A cell culture fluid is injected into the intravascular network space to dilute the vascular filling material, and the diluted vascular filling material is flushed out of the intravascular network space.

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