WO2022028565A1 - 仿生组织支架及其制备方法和应用 - Google Patents

仿生组织支架及其制备方法和应用 Download PDF

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WO2022028565A1
WO2022028565A1 PCT/CN2021/111119 CN2021111119W WO2022028565A1 WO 2022028565 A1 WO2022028565 A1 WO 2022028565A1 CN 2021111119 W CN2021111119 W CN 2021111119W WO 2022028565 A1 WO2022028565 A1 WO 2022028565A1
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parts
cartilage
layer
methacrylated
scaffold
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PCT/CN2021/111119
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English (en)
French (fr)
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陈慧敏
朱海林
汪争光
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华夏司印(上海)生物技术有限公司
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Priority claimed from CN202010785153.7A external-priority patent/CN113440651A/zh
Priority claimed from CN202010785145.2A external-priority patent/CN114053484A/zh
Application filed by 华夏司印(上海)生物技术有限公司 filed Critical 华夏司印(上海)生物技术有限公司
Priority to US18/005,963 priority Critical patent/US20230293306A1/en
Publication of WO2022028565A1 publication Critical patent/WO2022028565A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A—HUMAN NECESSITIES
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    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
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    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
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    • A61F2002/30001—Additional features of subject-matter classified in A61F2/28, A61F2/30 and subgroups thereof
    • A61F2002/30003—Material related properties of the prosthesis or of a coating on the prosthesis
    • A61F2002/30004—Material related properties of the prosthesis or of a coating on the prosthesis the prosthesis being made from materials having different values of a given property at different locations within the same prosthesis
    • A61F2002/30011—Material related properties of the prosthesis or of a coating on the prosthesis the prosthesis being made from materials having different values of a given property at different locations within the same prosthesis differing in porosity
    • A—HUMAN NECESSITIES
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    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
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    • A61F2002/3006—Properties of materials and coating materials
    • A61F2002/30062—(bio)absorbable, biodegradable, bioerodable, (bio)resorbable, resorptive
    • A—HUMAN NECESSITIES
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    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
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    • A61F2002/30985—Designing or manufacturing processes using three dimensional printing [3DP]

Definitions

  • the present invention relates to a bionic tissue scaffold and its preparation method and application.
  • articular cartilage lesions are almost involved in the early stages of pathological changes in almost all common clinical joint diseases. Due to the lack of blood vessels and lymphatic distribution in the cartilage tissue, the content of chondrocytes is low, the grandmother cells necessary for cell differentiation are lacking, and they are embedded in the thick extracellular matrix. The ability to repair itself is very poor, and even small cartilage defects are difficult to repair naturally.
  • tissue engineering technology provides a new idea and method for the treatment of articular cartilage injury.
  • Scaffolding material is one of the three major elements of tissue engineering technology. At present, many studies have used different biomaterials to prepare biomimetic cartilage scaffolds. However, the existing polymer material biomimetic cartilage scaffold has poor biocompatibility during use, and the absorption time is difficult to control; while the pure natural biomaterial biomimetic cartilage scaffold is too low in strength and difficult to prepare; the existing cartilage repair formula is easy to bone during use. At present, there is no clinical product with good cartilage repair effect.
  • tissue engineering osteochondral scaffolds is mainly divided into the following categories: 1) using scaffolds for bone but not cartilage, that is, high-density chondrocytes are directly planted on the bone scaffolds; 2) using two types of scaffolds suitable for bone and cartilage construction Scaffold material, respectively cultivated in vitro to form tissue engineered bone and cartilage, and then assembled the tissue engineered bone and cartilage parts into a tissue engineered osteochondral complex by bonding, or surgical suture, or sequential implantation; 3) Both bone and cartilage are An integrated single-layer scaffold using the same scaffold material; 4) an integrated double-layer scaffold constructed with two different scaffold materials for the osteochondral part.
  • the double-layered osteochondral scaffold has more excellent properties because its layered structure is designed according to the needs of bone and cartilage growth.
  • the current osteochondral biphasic scaffold also has the following problems:
  • the upper cartilage material has poor mechanical properties, absorbs water and deforms after implantation, and the degradation rate is fast;
  • the repaired cartilage is fibrocartilage instead of hyaline cartilage
  • the mechanical strength of the joint is not enough, and it is easy to separate; it is easy to hinder the penetration of the cartilage layer of the bone layer, resulting in obstacles to cell migration and nutrient delivery.
  • 3D bioprinting is an emerging technology for building tissues and organs, including organoids. In recent years, this technology has made great progress, but still has many limitations.
  • One of the toughest challenges is the precision and complexity of biomimetic tissue.
  • the technical methods of 3D printing include inkjet printing, laser-assisted printing or extrusion printing.
  • the hydrogel material prepared by bio-ink is too soft and has a long curing time, the shape maintenance accuracy is reduced, and it will collapse, making it difficult to maintain accurate printing effects.
  • 3D bioprinting needs to undergo a phase transition (photocuring) from a photosensitive hydrogel to a semi-solid cross-linked network via photo-initiated radical polymerization to form the corresponding biomaterial structure.
  • a better photocuring technology can effectively control/adjust the mechanical properties and degradation degree of the material, has good biocompatibility, and can enhance the elasticity of the printed structure and prolong the storage time on demand.
  • low-viscosity materials such as methacrylated gelatin, methacrylated sodium alginate, etc.
  • the light intensity and illumination time are not easy to accurately adjust, which makes the printing structure difficult. Hardness and strength are difficult to control, and it is difficult to form fine and complex structures.
  • the invention provides a preparation method of a bionic tissue scaffold, which comprises the following steps:
  • the definition of the hard polymer material is: 3D printing the polymer material with the goal of forming a cube bracket with a size of 10*10*10mm, and the size error of the actually formed bracket is within 10%.
  • Polymer materials can be called hard polymer materials.
  • the cube bracket described here is a standard for judging whether the polymer material is a hard polymer material, and does not limit the shape that the material can form. 3D printing using hard polymer materials, the shape is maintained well, and high-precision printing can be achieved.
  • Hard polymer materials that do not conform to the present invention include, for example, PEEK (polyetheretherketone), PEKK (polyetherketoneketone), PEI (polyetherimide) or PPSU (high performance medical grade plastic), all of which can be Printed by FDM, the stability is very good, but it is not suitable for use as a sacrificial material, and cannot be removed under normal conditions.
  • the sacrificial material is preferably biocompatible.
  • the "biocompatible" standard is: the biocompatibility test is carried out by the conventional method in the art, and the cell survival rate is above 75%.
  • the sacrificial material is preferably transparent or translucent.
  • the crosslinking cure is a photocrosslinking cure, the sacrificial material must be transparent or translucent.
  • the sacrificial material is preferably polylactic acid (PLA), polycaprolactone (PCL), polyethylene terephthalate-1,4-cyclohexane dimethanol (PETG), Polyvinyl alcohol (PVA) or synthetic photosensitive resin.
  • the synthetic photosensitive resin is preferably a polyacrylate photosensitive resin.
  • step S1 preferably, the pigment is mixed into the sacrificial material first, and then the colored sacrificial material is 3D printed to obtain a colored mold.
  • the effect of this is that when the mold is demolded in step S3, the disappearance of the color can be used as a monitoring indicator for the successful demoulding of the mold.
  • the 3D printing method can be a conventional printing method in the field that can realize precise microstructure, preferably an extrusion method (ie, a fusion deposition method) or a light curing method.
  • the light curing method may be Stereolithography (SLA), Digital Light Projection (DLP) or Liquid Crystal Display (LCD).
  • step S1 the shape, size and structure of the mold can be designed according to the required biomimetic tissue scaffold according to conventional methods in the art.
  • the hydrogel composition refers to a raw material composition for forming a hydrogel, including at least a gelable component and a gel medium.
  • the hydrogel composition may be a conventional hydrogel composition for biomimetic tissue scaffolds in the art.
  • each component in the hydrogel composition may exist in the form of a mixture, or may be separately packaged and mixed during use.
  • the gelling medium is generally not mixed with the gelable components, and when the gelling medium and the gelable components are separately packaged, the gelable components may be in powder, flake or Flocculent.
  • the gelatable component may be a conventional component in the art that can be cured to form a gel, and generally includes a natural gelatable component and/or a synthetic gelatable component.
  • the natural gelatable components can be conventional in the art, and preferably include natural proteins, natural protein modifications, natural protein degradation products, modifications of natural protein degradation products, natural polysaccharides, natural polysaccharide modifications, and natural polysaccharide degradation products. and one or more of modifications of natural polysaccharide degradants.
  • the natural protein includes one or more of various hydrophilic animal and vegetable proteins, water-soluble animal and vegetable proteins, type I collagen, type II collagen, serum protein, silk fibroin and elastin.
  • the native protein degradants preferably include gelatin (Gel) or polypeptides.
  • the modified product of the natural protein degradation product is preferably a methacrylylated natural protein degradation product, more preferably methacrylated gelatin (GelMA).
  • the natural polysaccharides include hyaluronic acid (HA), carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate (CS), one or more of ethylene glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan and chitosan quaternary ammonium salt, preferably hyaluronic acid acid (HA) and/or chondroitin sulfate (CS).
  • the natural polysaccharide modification is preferably a methacrylylated natural polysaccharide, such as methacrylated hyaluronic acid (HAMA) or methacrylated chondroitin sulfate (CSMA).
  • the synthetic gelable component can be conventional in the art, and preferably includes two-arm or multi-arm polyethylene glycol diacrylate, polyethylene imine, synthetic polypeptide, polyacrylic acid, polymethacrylic acid, polyacrylic acid One or more of ester, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl alcohol and polyvinylpyrrolidone.
  • the gelatinable components include methacrylated gelatin (GelMA), methacrylated collagen, methacrylated elastin, methacrylated hyaluronic acid (HAMA), methyl methacrylate Acrylated Chondroitin Sulfate (CSMA), Methacrylated Sodium Alginate, Methacrylated Heparin, Gelatin, Collagen, Elastin, Hyaluronic Acid, Chondroitin Sulfate, Heparin, and Sodium Alginate (Alg) one or a combination of several.
  • GelMA methacrylated gelatin
  • HAMA methacrylated hyaluronic acid
  • CSMA methyl methacrylate Acrylated Chondroitin Sulfate
  • Methacrylated Sodium Alginate Methacrylated Heparin
  • Gelatin Collagen
  • Elastin Hyaluronic Acid
  • Chondroitin Sulfate Heparin
  • Sodium Alginate Alg
  • the gelatinable component preferably comprises one of gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA) and chondroitin sulfate methacrylate (CSMA). one or more.
  • the biomimetic tissue scaffold is generally a cartilage scaffold or a bone scaffold.
  • the biomimetic tissue scaffold is a cartilage scaffold
  • the gelable components include sodium alginate (Alg) and methacrylated gelatin (GelMA).
  • the biomimetic tissue scaffold is a cartilage scaffold
  • the gelable components include methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA).
  • the biomimetic tissue scaffold is a cartilage scaffold
  • the gelable components include methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA) and methyl methacrylate Acryloyl Chondroitin Sulfate (CSMA).
  • the biomimetic tissue scaffold is a bone scaffold
  • the gelable components include sodium alginate (Alg), methacrylated gelatin (GelMA) and hydroxyapatite (HAp ).
  • the biomimetic tissue scaffold is a nerve conduit scaffold
  • the gelable components include sodium alginate and methacrylated gelatin.
  • the biomimetic tissue scaffold is a skin scaffold
  • the gelable components include methacrylated collagen and methacrylated gelatin.
  • the biomimetic tissue scaffold is a muscle scaffold
  • the gelable components include methacrylated hyaluronic acid and methacrylated gelatin.
  • the gel medium may be conventional in the art, preferably one or more of purified water, physiological saline, cell culture medium, calcium salt solution and phosphate buffer solution (PBS solution).
  • physiological saline is a 0.9% NaCl aqueous solution.
  • the cell culture medium can be a conventional cell culture medium in the art, such as DMEM, DMEM/F12, RPMI 1640 and other common culture medium.
  • the phosphate buffer solution can be conventional in the art, and the pH of the phosphate buffer solution is preferably 7.4.
  • the methacrylated gelatin may be conventional in the art, commercially available, and may also be obtained by methacrylating gelatin (Gel) by using a conventional method in the art.
  • the degree of methacrylation of the methacrylated gelatin may be 30% to 100%, preferably 40% to 80%.
  • the degree of methacrylation of the methacrylated gelatin is calculated by hydrogen nuclear magnetic resonance ( 1 H NMR), specifically: selecting the integral area of the standard peak of phenylalanine (7.1-7.4 ppm) as 1.
  • the degree of methacrylation of GelMA (the peak area of the lysine signal of Gel at 2.8-2.95ppm-the peak area of the lysine signal of GelMA at 2.8-2.95ppm)/the lysine signal of Gel at Peak area at 2.8 to 2.95 ppm*100%.
  • the methacrylated hyaluronic acid may be conventional in the art, commercially available, or may be obtained by methacrylating hyaluronic acid (HA) by using a conventional method in the art.
  • the molecular weight of the methacrylated hyaluronic acid may be 1-2000 kDa, preferably 100-1000 kDa, more preferably 500-950 kDa, more preferably 890-950 kDa.
  • the degree of methacrylation of the methacrylated hyaluronic acid may be 20% to 60%, preferably 30% to 50%.
  • the degree of methacrylation of the methacrylated hyaluronic acid is calculated using hydrogen nuclear magnetic resonance (1H NMR), specifically:
  • the methacrylated chondroitin sulfate can be conventional in the art, commercially available, and can also be obtained by methacrylating chondroitin sulfate (CS) by using a conventional method in the art.
  • the molecular weight of the methacrylated chondroitin sulfate may be 10-70 kDa, preferably 30-50 kDa.
  • the degree of methacrylation of the methacrylated chondroitin sulfate may be 20%-60%, preferably 30%-50%.
  • the degree of methacryloylation of the chondroitin methacrylate sulfate is calculated by using hydrogen nuclear magnetic resonance ( 1 H NMR), specifically:
  • Degree of methacrylation of CSMA peak area of methacrylamide-vinyl at 5.6 ppm/peak area of N-acetylglucose at 1.9 ppm*100%.
  • the hydrogel composition when the gelable component is a photosensitive gelable component, the hydrogel composition further includes a photoinitiator.
  • the photosensitive gelable ingredient is mixed with the gelling medium before the photoinitiator is added. The purpose of this is to dissolve the gelable components in the gel medium first, so as to facilitate the stable preservation of the hydrogel composition, and temporarily add the photoinitiator during use to avoid light during storage. The presence of the initiator results in some degree of crosslinking of the hydrogel composition.
  • the photoinitiator can be a conventional photoinitiator in the field, preferably a blue light initiator, an ultraviolet light initiator or a green light initiator;
  • the blue light initiator is preferably phenyl-2 ,4,6-trimethylbenzoylphosphonate lithium (LAP), riboflavin, flavin mononucleotide, eosin Y or terpyridine ruthenium chloride/sodium persulfate (Ru/SPS);
  • the initiator is preferably 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959).
  • the hydrogel composition may further include a thickening agent.
  • the thickener can be conventional in the art, preferably polyethylene oxide (PEO), polyethylene glycol (PEG), sodium alginate (Alg), hyaluronic acid, polyvinylpyrrolidone, gum arabic, One or more of gellan gum and xanthan gum.
  • the hydrogel composition may further include synthetic photosensitive materials.
  • the synthetic photosensitive material can be conventional in the art, preferably including polyethylene glycol acrylate (PEGDA), polyacrylic acid, polymethacrylic acid, polyacrylate, polymethacrylate, polyacrylamide and polymethylmethacrylate. one or more of acrylamides.
  • the synthetic photosensitive material is preferably polyethylene glycol acrylate.
  • the method of cross-linking and curing may include one or more of physical cross-linking and curing, chemical cross-linking and curing, and photo-cross-linking and curing; preferably, it includes photo-cross-linking and curing.
  • the cross-linking and curing can be performed by a conventional method in the art according to the properties of the gelable component.
  • the physical cross-linking and curing can be performed by a conventional method in the art, for example, the self-assembly curing of collagen at about 37°C.
  • the chemical cross-linking and curing can be carried out by conventional methods in the art, for example, the methacrylated material is catalyzed by ammonium persulfate to form a gel, or, sodium alginate is cross-linked with a divalent metal cation to form a gel colloid.
  • the photocrosslinking and curing can be carried out under light irradiation by a conventional method in the art; preferably, the photocrosslinking is carried out under light irradiation with a wavelength of 365-405 nm and an intensity of 5-50 mW/cm 2 ; more Preferably, the photo-crosslinking is carried out under the irradiation of light with a wavelength of 405 nm and an intensity of 10 mW/cm 2 .
  • the crosslinking curing is photocrosslinking curing.
  • the solvent may be selected according to the characteristics of the sacrificial material forming the mold, and the solvent may dissolve the mold.
  • the solvent is preferably dichloromethane, chloroform, tetrahydrofuran, 1,4-dioxane, purified water, physiological saline, calcium salt solution, phosphate buffered solution (PBS) or culture medium.
  • step S4 the freeze-drying time is preferably 8 to 24 hours; the pre-cooling step is preferably performed before the freeze-drying; the pre-cooling temperature is preferably -20°C, and the pre-cooling The time is preferably 1 to 3 hours.
  • the present invention also provides a biomimetic tissue scaffold, which is prepared according to the preparation method of the biomimetic tissue scaffold.
  • the biomimetic tissue scaffold can be biomimetic to biomimetic tissues conventional in the art, such as cartilage, bone, nerve conduit, ligament, muscle, breast, fat, skin, heart, liver, spleen, lung, kidney, pancreas, Stomach, intestine, bladder, blood vessels, etc.
  • the preparation method of the bionic tissue scaffold described in the present invention is an indirect 3D printing method, also known as a 3D printing demoulding method or a 3D engineering method.
  • the obtained biomimetic tissue scaffold has a microscopic fine grid-like structure (through structure), interconnected pores, adjustable porosity (up to 30% to 70%), and a large specific surface area (to form a scaffold with a side length of 10mm and a height of 3mm).
  • the surface area formed by the 3D printing through-hole structure (filling rate is 50%, the layer height is 0.2mm, and the nozzle size is 0.25mm) is 3200mm 2
  • the scaffold of the same size is formed by infusion, with a surface area of 320mm 2 , with a porous scaffold structure Compared with the non-porous structure, the surface area is increased by 10 times).
  • the present invention particularly provides an osteochondral scaffold, which includes a cartilage layer, an adhesion layer and a bone layer, and two sides of the adhesion layer are respectively connected with the cartilage layer and the bone layer; the cartilage layer, the One or more of the adhesion layer and the bone layer are porous structures.
  • the cartilage layer, the adhesion layer and the bone layer are all porous structures; more preferably, the pores of the cartilage layer, the pores of the adhesion layer and the bone The pores of the layers are connected.
  • the holes of the cartilage layer, the holes of the adhesion layer and the holes of the bone layer may be completely aligned or not completely aligned, and are preferably completely aligned.
  • the hole diameter of the cartilage layer and/or the bone layer is 50-350 ⁇ m, preferably 200-280 ⁇ m, for example, 250 ⁇ m;
  • the pores of the cartilage layer are of equal diameter.
  • the pore sizes of both the cartilage layer and the bone layer are selected to be suitable for cell capture and cell growth.
  • the holes of the cartilage layer and/or the bone layer are preferably distributed in a vertical cross arrangement.
  • the porosity of the cartilage layer and/or the bone layer is 20%-70%, preferably 40%-60%, such as 50%.
  • the porosity of the osteochondral scaffold may be 20%-70%, preferably 40%-60%, such as 50%.
  • the adhesion layer refers to the transition layer between the cartilage layer and the bone layer, which can realize the connection between the cartilage layer and the bone layer, and is not necessarily connected by bonding.
  • the adhesive layer does not cover or partially covers the pores of the bone layer and/or the cartilage layer. That is, the adhesive layer only covers part or all of the non-porous areas of the bone layer and the cartilage layer, so as to ensure that the adhesion layer does not block the pores of the bone layer and the cartilage layer.
  • the holes in the adhesion layer can be consistent with the holes in the cartilage layer and the holes in the bone layer, so as to ensure that the three layers pass through.
  • the shape of the osteochondral scaffold is not particularly limited, and in use, the osteochondral scaffold can be cut according to the size of the defect site.
  • the osteochondral scaffold is cylindrical.
  • the diameter of the cylinder may be 2-30 mm, preferably 2-20 mm, more preferably 3-10 mm; the height of the cylinder may be 2-10 mm, preferably 3-6 mm.
  • the osteochondral scaffold is a rectangular parallelepiped.
  • the bottom surface of the cuboid can be a square, and the side length of the square can be 2-30mm, preferably 2-20mm, more preferably 3-10mm; the height of the cuboid is preferably 2-10mm, More preferably, it is 3 to 6 mm.
  • the height ratio of the bone layer and the cartilage layer may be 1:(0.1-1), preferably 1:(0.2-0.5).
  • the height of the adhesive layer may be 5 ⁇ m ⁇ 2 mm, preferably 0.1 ⁇ 2 mm, more preferably 0.5 ⁇ 1 mm.
  • the material of the cartilage layer can be a conventional cartilage layer material in the art, preferably a hydrogel material.
  • the hydrogel material may be one or more of a single network hydrogel material, an interpenetrating network hydrogel material and a composite cross-linked hydrogel material.
  • a hydrogel material formed by a single cross-linking method is called a single network hydrogel material.
  • a hydrogel material formed by two or more cross-linking methods is called an interpenetrating network hydrogel material, or a double network hydrogel material. It is formed by compound cross-linking of multiple gelable components in the same cross-linking method, which is called compound cross-linked hydrogel material.
  • the hydrogel material is preferably a photocrosslinked hydrogel material, more preferably a composite photocrosslinked hydrogel material.
  • the cartilage layer preferably further carries a cartilage promoting component.
  • the cartilage-promoting component may include biologically active factors and/or cells.
  • the biologically active factor preferably includes transforming growth factor TGF ⁇ or TGF ⁇ .
  • the cells may include autologous or allogeneic chondrocytes, mesenchymal stem cells, embryonic stem cells, or induced pluripotent stem cells.
  • the material of the bone layer can be a conventional medical polymer material in the field, preferably polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA) or polycaprolactone (PCL).
  • PLA polylactic acid
  • PLGA polylactic acid-co-glycolic acid
  • PCL polycaprolactone
  • the material of the bone layer can also be a hydrogel material, and the hydrogel material is as described above.
  • the bone layer preferably also carries a bone-promoting component.
  • the bone-promoting component may include one or more of biologically active inorganic materials, biologically active factors and cells.
  • the bioactive inorganic material preferably includes one or more of hydroxyapatite, calcium phosphate, calcium carbonate and bioactive glass.
  • the mass percentage of the biologically active inorganic material in the bone layer may be 0.1wt%-70wt%, preferably 1wt%-50wt%, more preferably 2.5wt%-30wt%.
  • the biologically active factors preferably include transforming growth factors TGF ⁇ , TGF ⁇ , bone morphogenetic proteins BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP- 9, and one or more of cartilage-inducing compounds (eg, KGN, etc.).
  • the cells may include autologous or allogeneic bone cells, mesenchymal stem cells, embryonic stem cells, or induced pluripotent stem cells.
  • the material of the adhesive layer can be a hydrogel material, and the hydrogel material is as described above.
  • the adhesive layer may be formed from conventional medical glues in the art.
  • the medical glue can be selected from, for example, Compaq, Greensea, Golden Elephant, Hainuo, Shuangyi, Morphine, 3M, Qilekang, Fu Aile, IDEALPLAST, Kaiyan or Offit.
  • the cartilage layer, the adhesion layer and the bone layer are all hydrogel materials. At this time, the gelatable components in the cartilage layer, the adhesion layer, and the bone layer are gradually changed in concentration.
  • the present invention also provides a preparation method of the osteochondral scaffold, which comprises the following steps: connecting the bone layer and the cartilage layer, and forming an adhesion layer at the connection; the cartilage layer, the adhesion layer and the bone layer One or more of them are porous structures.
  • the preparation method of the cartilage layer can be conventional in the art, generally prepared by using a hydrogel composition as a raw material through cross-linking and curing.
  • a hydrogel composition as a raw material through cross-linking and curing.
  • the hydrogel composition and the method of cross-linking and curing are as described above.
  • the hydrogel composition for the cartilage layer includes the following components in parts by mass: 1-50 parts of methacrylated gelatin, methacrylated hyaluronate 0 to 30 parts of acid, 0 to 30 parts of methacrylated chondroitin sulfate, 0.01 to 1 part of photoinitiator and gel medium.
  • the dosage of the methacrylated gelatin is preferably 1-30 parts, more preferably 1-20 parts, more preferably 2-15 parts, more preferably 5-15 parts, such as 8 parts servings, 10 servings or 12 servings.
  • the dosage of the methacrylated hyaluronic acid is preferably 0.1-20 parts, more preferably 0.5-10 parts, more preferably 1-3 parts, such as 1.5 parts or 2 parts.
  • the dosage of the chondroitin methacryloyl sulfate is preferably 0.1-20 parts, more preferably 0.5-20 parts, more preferably 0.5-5 parts, more preferably 1-3 parts, For example 1 serving, 2 servings, 2.5 servings or 3 servings.
  • the mass ratio of the methacrylated gelatin and the methacrylated hyaluronic acid may be (1-30): (0.5-10), preferably (2-15): (1 ⁇ 3), such as 5:2.
  • the mass ratio of the methacrylated gelatin, the methacrylated hyaluronic acid and the methacrylated chondroitin sulfate may be (1-30):(0.5-10):( 0.5-20), preferably (2-15):1:(1-3), for example 10:1:3, 5:2:2, 15:1:1 or 8:1:3.
  • the amount of the gel medium can be conventional in the field, preferably, in the hydrogel composition: 5% to 30% of methacrylated gelatin (GelMA), methacrylated hyaluronate Acid (HAMA) 0.5-2%, chondroitin methacryloyl sulfate (CSMA) 0.1%-5%, photoinitiator 0.01-1%; the percentages are the mass of components contained per 100mL of gel medium (g) .
  • GelMA methacrylated gelatin
  • HAMA methacrylated hyaluronate Acid
  • CSMA chondroitin methacryloyl sulfate
  • the hydrogel composition of the cartilage layer may further include a thickening agent, and the amount of the thickening agent is preferably 0.1-25 parts.
  • the thickener is as previously described.
  • the dosage of the sodium alginate is preferably 0.5-2 parts.
  • the thickener includes hyaluronic acid, the amount of the hyaluronic acid used is preferably 0.5-2 parts.
  • the thickener includes polyvinylpyrrolidone, the dosage of the polyvinylpyrrolidone is preferably 2-10 parts.
  • the thickener includes gum arabic, the amount of the gum arabic used is preferably 0.1-25 parts.
  • the thickener includes gellan gum the amount of the gellan gum is preferably 0.1-2 parts.
  • the thickener includes xanthan gum, the amount of the xanthan gum is preferably 0.1-5 parts.
  • the hydrogel composition of the cartilage layer may further include a synthetic photosensitive material.
  • the amount of the synthetic photosensitive material is preferably 5-30 parts.
  • the synthetic photosensitive material is as described above.
  • the hydrogel composition of the cartilage layer includes the following components in parts by mass: 5-15 parts of methacrylated gelatin (GelMA), methacrylated hyaluronate Acid (HAMA) 0.5-2 parts, and photoinitiator 0.1-0.5 parts.
  • the hydrogel composition of the cartilage layer includes the following components in parts by mass: 5-15 parts of methacrylated gelatin (GelMA), methacrylated hyaluronate 0.5-2 parts of acid (HAMA), 0.5-3 parts of chondroitin methacryloyl sulfate (CSMA), and 0.1-0.5 parts of photoinitiator.
  • the hydrogel composition of the cartilage layer comprises the following components in parts by mass: 5-15 parts of methacrylated gelatin (GelMA), 1 part of sodium alginate (Alg) ⁇ 2 parts, and 0.1-0.5 parts of photoinitiator.
  • the hydrogel composition of the cartilage layer comprises the following components in parts by mass: 5 parts of methacrylated gelatin (GelMA), methacrylated hyaluronate 2 parts of acid (HAMA), 2 parts of chondroitin methacryloyl sulfate (CSMA), and 0.25 parts of photoinitiator.
  • the hydrogel composition of the cartilage layer includes the following components in parts by mass: 10 parts of methacrylated gelatin (GelMA), methacrylated hyaluronate 1 part of acid (HAMA), 3 parts of chondroitin methacryloyl sulfate (CSMA), and 0.25 part of photoinitiator.
  • the hydrogel composition of the cartilage layer comprises the following components in parts by mass: 15 parts of methacrylated gelatin (GelMA), methacrylated hyaluronate 1 part of acid (HAMA), 1 part of chondroitin methacryloyl sulfate (CSMA), and 0.25 part of photoinitiator.
  • the hydrogel composition of the cartilage layer comprises the following components in parts by mass: 8 parts of methacrylated gelatin (GelMA), methacrylated hyaluronate 1 part of acid (HAMA), 3 parts of chondroitin methacryloyl sulfate (CSMA), and 0.25 part of photoinitiator.
  • the hydrogel composition of the cartilage layer comprises the following components in parts by mass: 8 parts of methacrylated gelatin (GelMA), methacrylated hyaluronate 1 part of acid (HAMA), 3 parts of chondroitin methacrylate sulfate (CSMA), 2 parts of sodium alginate, and 0.25 part of photoinitiator.
  • the hydrogel composition of the cartilage layer comprises the following components in parts by mass: 8 parts of methacrylated gelatin (GelMA), methacrylated hyaluronate 1 part acid (HAMA), 3 parts chondroitin methacryloyl sulfate (CSMA), 10 parts PEGDA, and 1 part photoinitiator.
  • GelMA methacrylated gelatin
  • HAMA methacrylated hyaluronate 1 part acid
  • CSMA chondroitin methacryloyl sulfate
  • PEGDA photoinitiator
  • the hydrogel composition of the cartilage layer comprises the following components in parts by mass: 5 parts of methacrylated gelatin (GelMA), methacrylated hyaluronate 2 parts of acid (HAMA), and 0.5 part of photoinitiator.
  • the hydrogel composition of the cartilage layer includes the following components in parts by mass: 5 parts of methacrylated gelatin (GelMA), 2 parts of sodium alginate, and light 0.5 part of initiator.
  • the preparation method of the cartilage layer may be a perfusion method, which includes the following steps: pouring the hydrogel composition of the cartilage layer into a cartilage mold for photo-crosslinking to obtain a solidified hydrogel;
  • the solidified hydrogel can be obtained by freeze-drying.
  • the cartilage mold can be designed according to the shape and size of the cartilage required by conventional methods in the art.
  • the preparation method of the cartilage layer may be a direct 3D printing method, which includes the following steps: 3D printing the hydrogel composition of the cartilage layer and photocrosslinking to obtain a solidified hydrogel;
  • the solidified hydrogel is obtained by freeze-drying.
  • the preparation method of the cartilage layer includes: printing the hydrogel composition with an extrusion 3D printer, and curing it with blue light, wherein the temperature of the heat preservation is 30-37° C., and the temperature of the printing environment is 22 ⁇ 25°C, the printing pressure is 20 ⁇ 40PSI, the printing speed is 4 ⁇ 8mm/s, the filling rate is 40% ⁇ 60%, and the light intensity is 5 ⁇ 20mW/cm 2 .
  • the preparation method of the cartilage layer is preferably the preparation method of the aforementioned bionic tissue scaffold, that is, the 3D printing demoulding method, which specifically includes the following steps:
  • the sacrificial material, the hard polymer material, the 3D printing method, the operation and conditions of the cross-linking, the solvent, and the freeze-drying are all as described above.
  • the preparation method of the cartilage layer preferably further includes the step of loading cartilage promoting components.
  • the method of loading the cartilage-promoting ingredient can be routine in the art.
  • the cartilage promoting ingredient is as described above.
  • the loading method of the bioactive factor is generally to soak the cartilage layer in a bioactive factor solution, wherein the concentration of the bioactive factor solution may be 1 ⁇ g/mL ⁇ 200 ⁇ g/mL, preferably 5 ⁇ g/mL to 100 ⁇ g/mL.
  • the preparation method of the bone layer may be conventional in the field, and preferably the material of the bone layer is 3D printed.
  • the material of the bone layer is as described above.
  • the parameters of the 3D printing can be selected according to the structure and material of the bone layer using conventional methods in the art.
  • the 3D printing is preferably performed using a fused deposition 3D printer.
  • the preparation method of the bone layer further includes the step of loading a bone-promoting component.
  • the method for loading the bone-promoting components can be conventional in the art, for example: grinding the material of the bone layer into powder and then incorporating the bone-promoting component; or, dissolving the material of the bone layer in a solvent and then adding The bone-promoting component may be used, and then the solvent may be volatilized.
  • the bone-promoting ingredients are as described above.
  • the solvent may be an organic solvent.
  • the loading method of the biologically active factor is generally to soak the bone layer in a biologically active factor solution, wherein the concentration of the biologically active factor solution may be 1 ⁇ g/mL ⁇ 200 ⁇ g/mL, preferably 5 ⁇ g/mL to 100 ⁇ g/mL.
  • the bone layer is provided by 3D printing PLA loaded with hydroxyapatite using a fused deposition 3D printer, wherein the temperature of the print head is 210° C. and the temperature of the platform is 50-60° C. , the printing speed is 50 ⁇ 60mm/s, and the filling rate is 40% ⁇ 60%.
  • the bone layer is provided by 3D printing PCL loaded with tricalcium phosphate using a fused deposition 3D printer, wherein the printing temperature is 140-150° C., and the printing speed is 50-60 mm. /s, the filling rate is 40% to 60%.
  • the bone layer is provided by 3D printing GelMA loaded with tricalcium phosphate by using an extrusion light-curing 3D printer, wherein the temperature of the holding temperature is 30-35°C, and the temperature of the printing environment is 22 ⁇ 25°C, the printing pressure is 20 ⁇ 40PSI, the printing speed is 4-8mm/s, the filling rate is 40-60%, and the light intensity is 5 ⁇ 50mW/cm 2 .
  • the preparation method of the bone layer can also be prepared by using the hydrogel composition as a raw material through cross-linking and curing.
  • the hydrogel composition and the method of cross-linking and curing are as described above.
  • the hydrogel composition of the bone layer includes the following components: 5%-30% of methacrylated gelatin (GelMA), 2.5%-50% of bioactive glass and a photoinitiator 0.01% to 1%; wherein the percentage is the mass (g) of the components contained in every 100 mL of the gel medium.
  • connection is generally to connect the bone layer and the cartilage through medical glue.
  • the operating conditions of the connection can be determined according to the use method of the medical glue used.
  • the medical glue is as described above.
  • the osteochondral scaffold adopts the aforementioned preparation method of the bionic tissue scaffold, that is, the 3D printing demoulding method, to perform osteochondral integral molding, which specifically includes the following steps:
  • the hydrogel composition of the bone layer comprises the following components: 5%-30% of methacrylated gelatin (GelMA), 2.5%-50% of bioactive glass and photoinitiated
  • the hydrogel composition of the adhesive layer includes the following components: 5% to 30% of methacrylated gelatin (GelMA), 10% to 20% of bioactive glass and 0.01% of photoinitiator % ⁇ 1%
  • the hydrogel composition of the cartilage layer comprises the following components: 5% ⁇ 30% of methacrylated gelatin (GelMA), 0.5% ⁇ 2% of methacrylated hyaluronic acid (HAMA) %, chondroitin methacryloyl sulfate (CSMA) 0.5% to 5%, and photoinitiator 0.01% to 1%; wherein, the percentage is the mass (g) of the components contained in every 100 mL of the gel medium.
  • the sacrificial material, the hard polymer material, the 3D printing method, the operation and conditions of the cross-linking, the solvent, and the freeze-drying are all as described above.
  • the present invention also provides an application of the osteochondral scaffold in repairing cartilage defects.
  • the cartilage defect may be a composite osteochondral defect or a pure cartilage defect.
  • the osteochondral defect can be located in the knee, hip, or shoulder joint.
  • the osteochondral scaffold can be used as follows: drilling down to the bone layer at the osteochondral composite defect, after reaching the bone marrow cavity, removing the excess bone layer matrix, and placing the osteochondral scaffold material Placed in the osteochondral composite defect.
  • the bone marrow flowing out of the bone layer is rich in a large amount of mesenchymal stem cells, and when the bone marrow-derived mesenchymal stem cells flow through the osteochondral scaffold material, the bone marrow-derived mesenchymal stem cells are captured in the scaffold.
  • the wound is sutured to complete the repair of the osteochondral composite defect.
  • the osteochondral scaffold can be used in the following manner: placing the cartilage layer of the osteochondral scaffold at the defect, and performing microfracture treatment at the cartilage defect until the bone marrow flows out. At this time, the bone marrow emerging from the bone layer is rich in mesenchymal stem cells, and when flowing through the osteochondral scaffold, the cells are trapped in the scaffold. After the operation is completed, the wound is sutured to complete the repair of the simple cartilage defect.
  • the osteochondral scaffold of the present invention has the following advantages: (1) the osteochondral scaffold of the present invention has a refined through-hole structure to ensure up-down and horizontal penetration, which is conducive to the scaffold fully capturing cells when filling osteochondral defects, and Conducive to the transport of nutrients and metabolic wastes, thereby facilitating defect repair. Further, by selecting appropriate cartilage layer materials and bone layer materials, stem cells can be induced to differentiate and grow into chondrocytes and bone cells. (2) The osteochondral scaffold of the present invention has an excellent adhesion layer structure, which can firmly connect the cartilage layer and the bone layer to achieve the purpose of integrated integration; the adhesion layer acts as a transition layer of the bone layer to prevent the ossification of the cartilage layer.
  • the osteochondral scaffold of the present invention can realize full-thickness repair of osteochondral.
  • the osteochondral scaffold of the present invention has a simple clinical operation mode and practicability, and provides a new and effective solution for the clinical repair of cartilage defects and osteochondral composite defects.
  • the present invention also provides a hydrogel composition for biomimetic cartilage scaffold, which comprises the following components in parts by mass: 1-50 parts of methacrylated gelatin (GelMA), methacrylated transparent 0.1-30 parts of acid (HAMA), 0.1-30 parts of chondroitin methacrylate (CSMA), 0.01-1 part of photoinitiator and gel medium.
  • GelMA methacrylated gelatin
  • HAMA methacrylated transparent 0.1-30 parts of acid
  • CSMA chondroitin methacrylate
  • 0.01-1 part of photoinitiator 0.01-1 part of photoinitiator and gel medium.
  • the amount of the methacrylated gelatin is preferably 1-20 parts, more preferably 5-15 parts, such as 8 parts, 10 parts or 12 parts.
  • the degree of methacrylation of the methacrylated gelatin may be 30% to 100%, preferably 40% to 80%.
  • the dosage of the methacrylated hyaluronic acid is preferably 0.1-10 parts, more preferably 0.5-10 parts, further more preferably 0.5-2 parts, such as 0.5 parts, 1 part , 1.5 servings.
  • the molecular weight of the methacrylated hyaluronic acid may be 1-8000 kDa, preferably 100-1000 kDa, more preferably 500-950 kDa.
  • the degree of methacrylation of the methacrylated hyaluronic acid may be 20% to 60%, preferably 30% to 50%.
  • the dosage of the chondroitin methacryloyl sulfate is preferably 0.1-10 parts, more preferably 0.5-10 parts, still more preferably 0.5-3 parts, such as 1 part, 2 parts or 2.5 servings.
  • the molecular weight of the methacrylated chondroitin sulfate may be 5-50 kDa, preferably 10-40 kDa.
  • the degree of methacrylation of the methacrylated chondroitin sulfate may be 20% to 60%, preferably 30% to 50%.
  • the quality of the methacrylated gelatin, the methacrylated hyaluronic acid and the methacrylated chondroitin sulfate is preferably (2-15): (0.5-5 ): (1-5), more preferably (2-15): 1: (1-3), eg 10:1:3, 5:2:2, 15:1:1 or 8:1:3 .
  • the types of the photoinitiators are as described above.
  • the amount of the photoinitiator is preferably 0.1 to 0.5 part, for example, 0.25 part.
  • the hydrogel composition for biomimetic cartilage scaffold includes the following components in parts by mass: 5-15 parts of methacrylated gelatin (GelMA), methacryloyl 0.5-10 parts of hyaluronic acid (HAMA), 0.5-10 parts of chondroitin methacryloyl sulfate (CSMA), and 0.1-0.5 parts of photoinitiator.
  • the hydrogel composition for biomimetic cartilage scaffold comprises the following components in parts by mass: 5 parts of methacrylated gelatin (GelMA), methacrylic acid 2 parts acylated hyaluronic acid (HAMA), 2 parts methacrylated chondroitin sulfate (CSMA), and 0.25 part photoinitiator.
  • the hydrogel composition for biomimetic cartilage scaffold comprises the following components in parts by mass: 10 parts of methacrylated gelatin (GelMA), methacrylic acid 1 part of acylated hyaluronic acid (HAMA), 3 parts of chondroitin methacrylate sulfate (CSMA), and 0.25 part of photoinitiator.
  • the hydrogel composition for biomimetic cartilage scaffold comprises the following components in parts by mass: 15 parts of methacrylated gelatin (GelMA), methacrylic acid 1 part of hyaluronic acid acylated (HAMA), 1 part of chondroitin methacryloyl sulfate (CSMA), and 0.25 part of photoinitiator.
  • the hydrogel composition for biomimetic cartilage scaffold comprises the following components in parts by mass: 8 parts of methacrylated gelatin (GelMA), methacrylic acid 1 part of acylated hyaluronic acid (HAMA), 3 parts of chondroitin methacrylate sulfate (CSMA), and 0.25 part of photoinitiator.
  • the hydrogel composition for biomimetic cartilage scaffold comprises the following components in parts by mass: 8 parts of methacrylated gelatin (GelMA), methacrylic acid 1 part of acylated hyaluronic acid (HAMA), 3 parts of chondroitin methacryloyl sulfate (CSMA), 2 parts of sodium alginate, and 0.25 part of photoinitiator.
  • the hydrogel composition for biomimetic cartilage scaffold comprises the following components in parts by mass: 8 parts of methacrylated gelatin (GelMA), methacrylic acid 1 part of hyaluronic acid acylated (HAMA), 3 parts of chondroitin methacryloyl sulfate (CSMA), 10 parts of PEGDA, and 1 part of photoinitiator.
  • GelMA methacrylated gelatin
  • HAMA methacrylic acid 1 part of hyaluronic acid acylated
  • CSMA chondroitin methacryloyl sulfate
  • PEGDA photoinitiator
  • the hydrogel composition for biomimetic cartilage scaffold may further include a thickening agent, and the amount of the thickening agent is preferably 0.1-25 parts.
  • the type of the thickener is as described above.
  • the dosage of the sodium alginate is preferably 1-2 parts.
  • the thickener includes hyaluronic acid the amount of the hyaluronic acid used is preferably 0.5-2 parts.
  • the thickener includes polyvinylpyrrolidone the dosage of the polyvinylpyrrolidone is preferably 2-10 parts.
  • the thickener includes gum arabic the amount of the gum arabic used is preferably 0.1-25 parts.
  • the thickener includes gellan gum, the amount of the gellan gum is preferably 0.1-2 parts.
  • the thickening agent includes xanthan gum the amount of the xanthan gum is preferably 0.1-1 part.
  • the hydrogel composition for biomimetic cartilage scaffold may further include synthetic photosensitive materials.
  • the amount of the synthetic photosensitive material is preferably 5-30 parts.
  • the types of the synthetic photosensitive materials are as described above.
  • the gel medium is as described above.
  • the amount of the gel medium can be conventional in the art, and preferably, in the hydrogel composition for biomimetic cartilage scaffolds: methacrylated gelatin (GelMA) 5%-20%, methacrylic acid Acylated hyaluronic acid (HAMA) 0.1% to 3%, chondroitin methacryloyl sulfate (CSMA) 0.1% to 5%, photoinitiator 0.01% to 1%; the percentages are per 100mL gel medium Component mass (g).
  • the present invention also provides a method for preparing a bionic cartilage scaffold, which is obtained by using the hydrogel composition for the bionic cartilage scaffold as a raw material and being cured by photocrosslinking.
  • the preparation method of the biomimetic cartilage scaffold is preferably the preparation method of the biomimetic tissue scaffold as described above, that is, the 3D printing demoulding method, which specifically includes the following steps:
  • the sacrificial material, the hard polymer material, the 3D printing method, the operation and conditions of the cross-linking, the solvent, and the freeze-drying are all as described above.
  • the preparation method of the biomimetic cartilage scaffold may be a perfusion method, and the operation of the perfusion method is as described above.
  • the preparation method of the bionic cartilage scaffold may be a direct 3D printing method, and the operation of the direct 3D printing method is as described above.
  • the photocrosslinking and curing are as described above.
  • the present invention also provides a biomimetic cartilage support, which is prepared by the preparation method of the biomimetic cartilage support.
  • the present invention also provides the hydrogel composition for biomimetic cartilage scaffold or the application of the biomimetic cartilage scaffold in osteochondral tissue engineering.
  • the hydrogel composition for the biomimetic cartilage scaffold provided by the present invention can prepare the biomimetic cartilage scaffold by methods such as perfusion or 3D printing, and the preparation method is simple and feasible.
  • the prepared biomimetic cartilage scaffold has the following advantages: (1) good biocompatibility; (2) degradable and replaced by regenerated cartilage; (3) inhibiting ossification into osteogenesis; (4) inducing cartilage formation; (5) 3D printing
  • the demolding method can obtain high-precision through-hole bionic cartilage scaffolds.
  • FIG. 1 is a drawing of the mold design of the cartilage scaffolds of Examples 1 to 3 of the present invention.
  • Example 2 is a microscope photograph of the mold of the cartilage scaffold of Example 1 of the present invention.
  • Example 3 is a microscope photograph of the cartilage scaffold of Example 1 of the present invention.
  • Example 4 is a camera photograph of the mold of the cartilage scaffold of Example 2 of the present invention.
  • FIG. 5 is a camera photograph of the cartilage scaffold of Example 2 of the present invention.
  • FIG. 6 is a drawing of the mold design of the bone scaffold according to Example 4 of the present invention.
  • FIG. 7 is a camera photograph of the mold of the bone scaffold of Example 4 of the present invention.
  • FIG. 8 is a camera photograph of the cured hydrogel of Example 4 of the present invention after rehydration.
  • Fig. 9 is the mold design drawing of the nerve conduit stent of the embodiment 5 of the present invention.
  • FIG. 10 is a real drawing of the mold printing of the nerve conduit stent of Example 5 of the present invention.
  • FIG. 11 is a diagram of the cured gel-mold composite of the nerve conduit stent of Example 5 of the present invention.
  • Example 12 is a picture of the catheter stent of Example 5 of the present invention after demolding.
  • FIG. 13 is a drawing of the mold design of the skin scaffold of Example 6 of the present invention.
  • FIG. 14 is a real drawing of the mold printing of the skin support of Example 6 of the present invention.
  • Example 15 is a diagram of the cured gel-mold composite of the skin scaffold of Example 6 of the present invention.
  • FIG. 16 is a picture of the skin stent of Example 6 of the present invention after demolding.
  • FIG. 17 is a drawing of the mold design of the muscle scaffold of Example 7 of the present invention.
  • FIG. 18 is a real drawing of the mold printing of the muscle scaffold of Example 7 of the present invention.
  • FIG. 19 is a diagram of the cured gel-mold composite of the muscle scaffold of Example 7 of the present invention.
  • FIG. 20 is a picture of the muscle scaffold of Example 7 of the present invention after demolding.
  • FIG. 21 is a microscope photograph of MSC cells cultured on the cartilage scaffold of Example 1 of the present invention.
  • FIG. 22 is a schematic structural diagram of the osteochondral scaffolds in Examples 8 to 11 of the present invention.
  • FIG. 23 is a camera photograph of the osteochondral scaffold of Example 8 of the present invention.
  • Example 24 is a microscope photograph of the osteochondral scaffold in Example 8 of the present invention.
  • FIG. 25 is a microscope photograph of MSC cells cultured on osteochondral scaffolds in Example 2 of the effect of the present invention.
  • Fig. 26 is a general observation photograph of the injured joint in group (a) in Example 3 of the effect of the present invention.
  • Figure 27 is a general observation photograph of the injured joint in group (b) in Example 3 of the effect of the present invention.
  • Figure 28 is a general observation photograph of the injured joint in group (c) in Example 3 of the effect of the present invention.
  • Fig. 29 is the change of the modulus of the hydrogel of the effect of the present invention 4 with time under illumination.
  • FIG. 30 is the stress-strain curve of the cured hydrogel scaffold of Example 5 of the effect of the present invention.
  • FIG. 31 shows the viability of cells after culturing in the solidified hydrogel of Example 6 of the effect of the present invention for 7 days (green staining of live cells, red staining of dead cells).
  • FIG. 32 is a general observation photograph in Example 7 of the effect of the present invention.
  • FIG. 33 is a view of the tissue section in Example 7 of the effect of the present invention.
  • the preparation method of cartilage scaffold comprises the following steps:
  • the obtained solidified hydrogel is placed in a -20° C. refrigerator to pre-cool for 2 hours, and then freeze-dried for 8 hours by a freeze dryer to obtain a cartilage scaffold (as shown in FIG. 3 ).
  • the preparation method of cartilage scaffold comprises the following steps:
  • the obtained solidified hydrogel is placed in a -20° C. refrigerator for pre-cooling for 2 hours, and then freeze-dried by a freeze dryer for 20 hours to obtain a cartilage scaffold (as shown in FIG. 5 ).
  • the preparation method of cartilage scaffold comprises the following steps:
  • the preparation method of bone scaffold it comprises the following steps:
  • the preparation method of nerve conduit stent it comprises the following steps:
  • the preparation method of skin support it comprises the following steps:
  • the obtained solidified hydrogel is placed in a -20°C refrigerator for 2 hours to be pre-cooled, and then freeze-dried by a freeze dryer for 20 hours; that is, a skin scaffold is obtained.
  • the preparation method of muscle scaffold it comprises the following steps:
  • mesenchymal stem cell MSCs were planted on the scaffold material, and then culture medium was added, and cultured at 37°C/5% CO 2 for 24 h. Before testing, the cell culture medium was aspirated and washed several times with PBS, followed by the addition of 1 mL of cell live/dead double staining reagent (10 ⁇ M calcein and 15 ⁇ M diethorphine dissolved in 5 mL PBS) at 37°C. Incubate with cells for 30 min, and then use confocal fluorescence microscope to observe the adhesion and survival of cells inside the scaffold material.
  • cell live/dead double staining reagent 10 ⁇ M calcein and 15 ⁇ M diethorphine dissolved in 5 mL PBS
  • Live cells showed calcein staining activity, emitting green fluorescence at 433 nm; dead cells were stained with ethidium bromide, emitting red fluorescence at 543 nm excitation. It can be seen from Fig. 21 that this type of scaffold material has good cytocompatibility and can grow into the through-hole structure of the scaffold material.
  • the integral area of the standard peak of phenylalanine (7.1-7.4 ppm) was selected as 1, and the reduction of the peak area of the lysine signal at 2.8-2.95 ppm before and after gelatin modification was calculated. Percentage, that is, the degree of methacrylation of the methacrylated gelatin is 65%.
  • HAMA methacrylated hyaluronic acid
  • the hydrogel composition is poured into a prefabricated cylindrical mold (diameter 5mm, height 3mm), and photocrosslinking is achieved under the irradiation of a light source with a wavelength of 405nm and an intensity of 10mW/ cm2 , and GelMA/HAMA photocrosslinking and curing can be obtained.
  • Hydrogel the prepared GelMA/HAMA photocrosslinked and solidified hydrogel was placed in a -20°C refrigerator for 2 hours, and then freeze-dried by a freeze dryer to obtain a GelMA/HAMA cartilage layer.
  • TGF ⁇ loading soak the above-mentioned GelMA/HAMA cartilage layer in 10 ⁇ g/mL TGF ⁇ solution, after fully adsorbing for 12 hours, place it in a -20°C refrigerator for 2 hours, and then freeze-dry it with a freeze dryer to obtain The cartilage layer loaded with TGF ⁇ was denoted as TGF ⁇ -GelMA/HAMA cartilage layer.
  • the HAP/PLA polymer material was printed by a fused deposition 3D printer (printing temperature: 210 °C; platform temperature: 50 °C; printing speed: 60 mm/s; filling rate: 50%, layer height: 0.1 mm) to obtain HAP /PLA bone layer.
  • the obtained HAP/PLA bone layer was cylindrical (5 mm in diameter, 3 mm in height), with a porosity of 50% and a pore diameter of 250 ⁇ m.
  • TGF ⁇ -GelMA/HAMA cartilage layer and HAP/PLA bone layer were connected with a medical glue golden elephant, and an adhesion layer was formed at the connection, and the thickness of the adhesion layer was about 100 ⁇ m, that is, TGF ⁇ -GelMA/HAMA-HAP/PLA bone layer was obtained.
  • the cartilage scaffold its structural schematic diagram is shown in Figure 22, from top to bottom are the cartilage layer 1, the adhesion layer 2 and the bone layer 3; the physical camera photo is shown in Figure 23; the microscopic structure is observed through a microscope, as shown in Figure 24.
  • the above-mentioned hydrogel composition was printed by an extrusion 3D printer, and cured by blue light (holding temperature: 37° C.; platform temperature: 22° C.; printing pressure: 20 PSI; printing speed: 5 mm/s; filling rate: 50%);
  • the prepared GelMA/HAMA photocrosslinked and solidified hydrogel was placed in a -20°C refrigerator for 2 h, and then freeze-dried by a freeze dryer to obtain a GelMA/HAMA cartilage layer.
  • the obtained GelMA/HAMA cartilage layer was cylindrical (5 mm in diameter and 1 mm in height), with a porosity of 50% and a pore diameter of 300 ⁇ m.
  • TGF ⁇ -GelMA/HAMA cartilage layer and HAP/PLA bone layer were connected with a medical glue golden elephant, and an adhesion layer was formed at the connection, and the thickness of the adhesion layer was about 100 ⁇ m, that is, TGF ⁇ -GelMA/HAMA-HAP/PLA bone layer was obtained.
  • the cartilage scaffold, the schematic diagram of its structure is shown in Figure 22.
  • the hydrogel composition is poured into a prefabricated mold, and photocrosslinking is realized under the irradiation of a light source with a wavelength of 405 nm and an intensity of 10 mW/cm 2 ; Then, the prepared composite photocrosslinked and cured hydrogel was placed in a -20 °C refrigerator for 2 h, and then frozen in a freeze dryer. Dry to obtain the Alg/GelMA cartilage layer.
  • MSCs mesenchymal stem cells
  • PCL polycaprolactone
  • TCP tricalcium phosphate
  • TCP/PCL polymer material The TCP/PCL polymer material is printed with a fused deposition 3D printer (printing temperature: 140°C; printing pressure: 40PSI; printing speed: 50mm/s; filling rate: 50 %) to obtain the TCP/PCL bone layer.
  • the obtained TCP/PCL bone layer was cylindrical (5 mm in diameter, 3 mm in height), with a porosity of 50% and a pore diameter of 300 ⁇ m.
  • the above-mentioned MSC-Alg/GelMA cartilage layer and the TCP/PCL bone layer were connected with the medical glue golden elephant, and the adhesion layer was formed at the connection, and the thickness of the adhesion layer was about 100 ⁇ m, that is, the MSC-Alg/GelMA-TCP/PCL bone layer was obtained.
  • the cartilage scaffold, the schematic diagram of its structure is shown in Figure 22.
  • the Alg/GelMA-TCP/PCL osteochondral scaffold was obtained.
  • the prepared Alg/GelMA composite photocrosslinked solidified hydrogel in a -20°C refrigerator for 2 hours, and then freeze-drying with a freeze dryer to obtain an Alg/GelMA cartilage layer.
  • the obtained Alg/GelMA cartilage layer was cylindrical (5 mm in diameter and 1 mm in height), with a porosity of 50% and a pore diameter of 250 ⁇ m.
  • MSCs mesenchymal stem cells
  • the above-mentioned MSC-Alg/GelMA cartilage layer and the TCP/PCL bone layer were connected with the medical glue golden elephant, and the adhesion layer was formed at the connection, and the thickness of the adhesion layer was about 100 ⁇ m, that is, the MSC-Alg/GelMA-TCP/PCL bone layer was obtained.
  • the cartilage scaffold, the schematic diagram of its structure is shown in Figure 22.
  • the Alg/GelMA-TCP/PCL osteochondral scaffold was obtained.
  • chondroitin sulfate methacrylate Dissolve chondroitin sulfate (10 g, 30 kDa) in 100 mL of deionized water, cool to 0-4°C, add 50 mL of methacrylic anhydride, and slowly drop Add 50mL of 5M NaOH aqueous solution, react for 24h, then pour the reaction solution into a dialysis bag (mWCO 7000), dialyze with deionized water for 2-3d, and freeze-dry to obtain chondroitin methacryloyl sulfate (9g).
  • mWCO 7000 dialysis bag
  • Hydrogel composition of cartilage layer weigh 20 g of methacrylated gelatin (GelMA), 1 g of methacrylated hyaluronic acid (HAMA), and 1 g of methacrylated chondroitin sulfate (CSMA), dissolved in deionized water at 50°C, adding 0.1 g of the initiator LAP to prepare a hydrogel of the cartilage layer, wherein the percentage is the mass (g) of the components contained in each 100 mL of the gel medium;
  • GelMA methacrylated gelatin
  • HAMA methacrylated hyaluronic acid
  • CSMA methacrylated chondroitin sulfate
  • transition layer Weigh 20 g of methacrylated gelatin (GelMA) and 10 g of bioactive glass, dissolve them in deionized water at 50°C, add 0.25 g of initiator LAP, The hydrogel is formulated into a transition layer; wherein the percentage is the mass (g) of the components contained in every 100 mL of the gel medium;
  • Hydrogel composition of bone layer weigh 20 g of methacrylated gelatin (GelMA) and 50 g of bioactive glass, dissolve in deionized water at 50°C, add 0.2 g of initiator LAP, The hydrogel is formulated into a bone layer; wherein the percentage is the mass (g) of the component contained per 100 mL of the gel medium.
  • GelMA methacrylated gelatin
  • bioactive glass dissolve in deionized water at 50°C, add 0.2 g of initiator LAP
  • the hydrogel composition of the bone layer, the adhesion layer and the cartilage layer is poured into the osteochondral scaffold mold in turn, and in-situ photocrosslinking is realized under the irradiation of a light source with a wavelength of 405 nm and an intensity of 10 mW/cm 2 to obtain photocrosslinking.
  • Joint-solidified hydrogel-osteochondral scaffold mold complex
  • the obtained osteochondral scaffold was a rectangular parallelepiped (the bottom surface was 30 mm*30 mm, the height was 3 mm), the porosity was 50%, and the pore diameter was 250 ⁇ m.
  • the mesenchymal stem cells were digested with trypsin, the cells were collected by centrifugation, and then the cell suspension was added dropwise to the above osteochondral scaffolds. Incubate for 24 h in the incubator. Before testing, the cell culture medium was aspirated and washed several times with PBS, followed by the addition of 1 mL of cell live/dead double-staining reagent (10 ⁇ M calcein and 15 ⁇ M diethorphine in 5 mL PBS) at 37°C. Incubate with cells for 30 min.
  • the adhesion and survival of cells in the osteochondral scaffold were observed by confocal fluorescence microscopy.
  • Live cells showed calcein staining activity, emitting green fluorescence at 433 nm; dead cells were stained with ethidium bromide, emitting red fluorescence at 543 nm excitation. It can be seen from FIG. 25 that the osteochondral scaffold of the present invention has good cytocompatibility and can grow into the through-hole structure of the scaffold material.
  • New Zealand male white rabbits were used, and a model of osteochondral composite defect was established in each rabbit. Before the experiment, they were randomly divided into groups according to their body weight (3 animals in each group): a: blank control group; b: bone layer scaffold (HAP/PLA) negative control group; c: osteochondral scaffold (GelMA/HAMA-HAP/PLA) group. During surgery, the scaffold was filled in the articular osteochondral complex defect of rabbits. After 12 weeks of operation, the rabbits in the experiment were sacrificed by intravenous injection of air, and the injured joints were extracted to evaluate the experimental repair effect. The gross observation photographs of the injured joint are shown in Figs. 26 to 28. Fig.
  • FIG. 26 is a blank control group, since no scaffold is placed, almost no new tissue can be seen.
  • Fig. 27 is the negative control group with only the bone layer scaffold, because the cartilage layer is missing, no new cartilage grows at all, and only the bone layer scaffold that has not been degraded is seen.
  • Figure 28 shows the osteochondral scaffold group. It can be seen that new tissue is formed where the osteochondral scaffold is implanted, and it has a similar appearance to the surrounding normal tissue and has a good repair effect.
  • methacrylated gelatin (GelMA: SR-3DP-0201), methacrylated hyaluronic acid (HAMA: SR-3DP-0301), methyl methacrylate Acrylated chondroitin sulfate (CSMA: SR-3DP-0401) and LAP were purchased from Huaxia Printing (Shanghai) Biotechnology Co., Ltd.
  • the degree of methacrylation of GelMA is 65%; the molecular weight of HAMA is 900 kDa, and the degree of methacrylation is 40%; the molecular weight of CSMA is 30 kDa, and the degree of methacrylation is 40%.
  • the cured hydrogel was placed in a -20°C refrigerator for 2 hours, and then freeze-dried for 8 hours by a freeze dryer to prepare a biomimetic cartilage scaffold of 8% GelMA/1% HAMA/5% CSMA.
  • the prepared solidified hydrogel was placed in a -20 °C refrigerator for 2 h, and then lyophilized for 20 h in a freeze dryer to prepare 8% GelMA/1% HAMA/3% CSMA/2% Alg biomimetic cartilage. bracket.
  • the maximum pressure before the cured hydrogel scaffold is broken is the ultimate stress, and the elastic modulus is calculated according to the slope of 15% to 20% of the stress-strain curve. It can be seen from FIG. 30 that the ultimate stress of the cured hydrogel scaffold is 170.8 kPa, and the elastic modulus is 123.7 kPa.
  • Figure 33 The histological analysis of Figure 33 also showed that chondrocyte lacuna was formed in the medial condyle defect, while no chondrocyte lacuna was found in the trochlear blank control; the cartilage matrix stained with safranine O was red, and the toluidine blue stained purple red, indicating that the internal New cartilage matrix was secreted in the condyle defect, and the formation of cancellous bone was observed under the cartilage; while the control trochlear had no cartilage matrix secretion and no cancellous bone formation.

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Abstract

本发明公开了一种仿生组织支架及其制备方法和应用。本发明的制备方法为3D打印脱模法，该制备方法可实现水凝胶材料的高精度3D工程制造。采用该制备方法获得的仿生组织支架具有微观精细网格状结构，孔隙连通，孔隙率高，比表面积大，尤其是仿生软骨支架、骨软骨支架，在关节软骨病变的治疗中有很大的应用价值。

Description

仿生组织支架及其制备方法和应用
本申请要求申请日为2020/8/6的中国专利申请2020107851452、申请日为2020/8/6的中国专利申请2020107851611和申请日为2020/8/6的中国专利申请2020107851537的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种仿生组织支架及其制备方法和应用。
背景技术
关节软骨病变作为一个基本的病理过程，几乎参与了所有临床常见的关节疾病病理变化早期阶段。由于软骨组织内缺乏血管和淋巴分布，软骨细胞含量少，缺少细胞分化所必须的祖母细胞且包埋于稠厚的细胞外基质中，迁徙难度大，无法有效移动到损伤部位参与修复，因此其自我修复能力非常差，即使是微小的软骨缺损也难以自然修复。组织工程技术的出现为关节软骨损伤的治疗提供了一种全新的思路和方法。
支架材料是组织工程技术的三大要素之一。目前已有很多研究用不同的生物材料制备仿生软骨支架。然而，现有的高分子材料仿生软骨支架在使用过程生物兼容性差，吸收时间难控制；而纯天然生物材料仿生软骨支架强度太低，且制备困难；现有软骨修复配方在使用过程中易骨化或纤维化，目前尚无软骨修复效果良好的临床产品。
目前组织工程骨软骨支架设计主要分为以下几类：1)骨采用支架而软骨不采用支架，即在骨支架上方直接种植上高密度的软骨细胞；2)采用适合骨、软骨构建的两种支架材料，分别在体外培养形成组织工程骨和软骨，然后粘合、或手术缝合、或顺序植入等方法将组织工程骨和软骨部分组装成组织工程骨软骨复合体；3)骨和软骨皆采用相同的支架材料的一体化单层支架；4)骨软骨部分分别采用两种不同的支架材料构建的一体化的双层支架。
双层骨软骨支架，由于其分层结构是根据骨和软骨生长的需要而设计的，因此具有更优良的特性。然而目前这种骨软骨双相支架也存在以下问题：
1)上层软骨材料力学性能差，植入体内后吸水变形，降解速率快；
2)修复后的软骨是纤维软骨而不是透明软骨；
3)新生软骨与周边软骨组织整合不良。已有的临床产品，通过灌注来修复骨软骨缺损，没有通孔结构，细胞无法进入支架内部，只在表面停留，修复效果不好；另外，还可 能长成瘢痕组织或者骨化成骨；
4)连接部力学强度不够，容易分离；容易阻碍骨层软骨层的贯通，导致细胞移行、营养物质输送障碍。
生物3D打印技术是构建组织和器官(包括类器官)的新兴技术。近几年，此项技术已取得了长足的进展，但是仍然具有很多局限性。其中一项最艰巨的挑战是对仿生组织的精度和复杂度的挑战。3D打印的技术方法包括喷墨式打印、激光辅助式打印或挤出式打印等，挤出式打印更适合生物3D打印；其中挤出式打印适用的生物墨水种类也最为丰富。但是，由于生物墨水制备的水凝胶材料太软、固化时间较长，形态维持精度下降，会坍塌，难以保持精准的打印效果。
一般来讲，生物3D打印需要经历一个从光敏水凝胶到通过光引发的自由基聚合反应实现的半固态交联网络的相变(光固化)，以形成相应的生物材料结构。较好的光固化技术可以有效控制/调节材料的力学性能和降解度，生物相容性好，并可按需增强打印结构的弹性和延长储存时间。而目前采用的光固化技术，低粘度的材料(如甲基丙烯酰化明胶、甲基丙烯酰化海藻酸钠等)不容易成型，光的强度和光照时间不容易精确调整，使得打印结构的硬度和强度难以控制，难以形成精细复杂结构。
发明内容
本发明提供一种仿生组织支架的制备方法，其包括以下步骤：
S1、将牺牲材料进行3D打印，得到模具；其中，所述牺牲材料为可溶于溶剂的硬质高分子材料；
S2、向所述模具中灌注水凝胶组合物，使其原位交联固化，得到固化水凝胶-模具复合体；
S3、脱模：用溶剂溶解所述固化水凝胶-模具复合体中的模具，得到固化水凝胶；
S4、将所得固化水凝胶冷冻干燥即得所述仿生组织支架。
本发明中，所述硬质高分子材料的定义为：以形成尺寸为10*10*10mm的立方体支架为目标对高分子材料进行3D打印，实际形成的支架的尺寸误差在10％以内，该高分子材料即可称为硬质高分子材料。此处所述立方体支架为判断高分子材料是否为硬质高分子材料的标准，并不对材料可形成的形状产生限制。采用硬质高分子材料进行3D打印，形态维持良好，可实现高精度打印。
目前行业内通常使用的常规牺牲材料，例如普朗尼克、卡波姆、明胶颗粒、蔗糖等，在3D打印过程中无法维持形态，不能实现精细打印。不符合本发明的硬质高分子材料例 如有PEEK(聚醚醚酮)、PEKK(聚醚酮酮)、PEI(聚醚酰亚胺)或PPSU(高性能医疗级塑料)，这些材料均可通过FDM方式打印，稳定性非常好，用作牺牲材料则不合适，常规条件无法去除。
本发明中，所述牺牲材料较佳地为生物相容的。所述“生物相容的”标准为：采用本领域常规的方法进行生物相容性测试，细胞存活率在75％以上。
本发明中，所述牺牲材料较佳地为透明的或半透明的。当所述交联固化为光交联固化时，所述牺牲材料须为透明的或半透明的。
本发明中，所述牺牲材料较佳地为聚乳酸(PLA)、聚己内酯(PCL)、聚对苯二甲酸乙二醇酯-1,4-环己烷二甲醇酯(PETG)、聚乙烯醇(PVA)或合成类光敏树脂。所述合成类光敏树脂较佳地为聚丙烯酸酯类光敏树脂。
本发明中，步骤S1中，较佳地，先将颜料混于所述牺牲材料中，然后将带颜色的牺牲材料进行3D打印，得到带颜色的模具。这样做的作用为，在步骤S3脱模时，颜色消失可以作为模具脱除成功的监控指标。
本发明中，步骤S1中，所述3D打印的方式可为本领域常规的可以实现精确细微结构的打印方式，较佳地为挤出方式(即熔融堆积方式)或光固化方式。所述光固化方式可为立体光固化成型技术(SLA)、数字光投影技术(DLP)或液晶显示技术(LCD)。
本发明中，步骤S1中，所述模具的形状、大小和结构可根据所需的仿生组织支架按照本领域常规的方法设计。
本发明中，所述水凝胶组合物是指形成水凝胶的原料组合物，至少包括可凝胶成分和凝胶介质。所述水凝胶组合物可为本领域常规的用于仿生组织支架的水凝胶组合物。
本发明中，所述水凝胶组合物中各组分可以混合物的形式存在，也可单独分装，在使用时混合。其中，所述凝胶介质一般不与所述可凝胶成分混合，当所述凝胶介质与所述可凝胶成分单独分装时，所述可凝胶成分可为粉状、片状或絮状。
其中，所述可凝胶成分可为本领域常规的可以固化形成凝胶的成分，一般包括天然可凝胶成分和/或合成可凝胶成分。
所述天然可凝胶成分可为本领域常规，较佳地包括天然蛋白、天然蛋白修饰物、天然蛋白降解物、天然蛋白降解物的修饰物、天然多糖、天然多糖修饰物、天然多糖降解物和天然多糖降解物的修饰物中的一种或多种。
所述天然蛋白包括各种亲水动植物蛋白、水溶性动植物蛋白、I型胶原蛋白、II型胶原蛋白、血清蛋白、丝素蛋白和弹性蛋白中的一种或多种。所述天然蛋白降解物较佳地包括明胶(Gel)或多肽。所述天然蛋白降解物的修饰物较佳地为甲基丙烯酰化天然蛋白 降解物，更佳地为甲基丙烯酰化明胶(GelMA)。
所述天然多糖包括透明质酸(HA)、羧甲基纤维素、甲基纤维素、羟乙基纤维素、羟丙基纤维素、海藻酸、葡聚糖、琼脂糖、肝素、硫酸软骨素(CS)、乙二醇壳聚糖、丙二醇壳聚糖、壳聚糖乳酸盐、羧甲基壳聚糖和壳聚糖季铵盐中的一种或多种，较佳地为透明质酸(HA)和/或硫酸软骨素(CS)。所述天然多糖修饰物较佳地为甲基丙烯酰化天然多糖，例如甲基丙烯酰化透明质酸(HAMA)或甲基丙烯酰化硫酸软骨素(CSMA)。
其中，所述合成可凝胶成分可为本领域常规，较佳地包括两臂或多臂聚乙二醇双丙烯酸酯、聚乙烯亚胺、合成多肽、聚丙烯酸、聚甲基丙烯酸、聚丙烯酸酯、聚甲基丙烯酸酯、聚丙烯酰胺、聚甲基丙烯酰胺、聚乙烯醇和聚乙烯吡咯烷酮中的一种或多种。
较佳地，所述可凝胶成分包括甲基丙烯酰化明胶(GelMA)、甲基丙烯酰化胶原、甲基丙烯酰化弹力蛋白、甲基丙烯酰化透明质酸(HAMA)、甲基丙烯酰化硫酸软骨素(CSMA)、甲基丙烯酰化海藻酸钠、甲基丙烯酰化肝素、明胶、胶原、弹力蛋白、透明质酸、硫酸软骨素、肝素和海藻酸钠(Alg)中的一种或几种的组合。
本发明中，所述可凝胶成分较佳地包括甲基丙烯酰化明胶(GelMA)、甲基丙烯酰化透明质酸(HAMA)和甲基丙烯酰化硫酸软骨素(CSMA)中的一种或多种。此时，所述仿生组织支架一般为软骨支架或骨支架。
在本发明一较佳的实施方式中，所述仿生组织支架为软骨支架，所述可凝胶成分包括海藻酸钠(Alg)和甲基丙烯酰化明胶(GelMA)。
在本发明一较佳的实施方式中，所述仿生组织支架为软骨支架，所述可凝胶成分包括甲基丙烯酰化明胶(GelMA)和甲基丙烯酰化透明质酸(HAMA)。
在本发明一较佳的实施方式中，所述仿生组织支架为软骨支架，所述可凝胶成分包括甲基丙烯酰化明胶(GelMA)、甲基丙烯酰化透明质酸(HAMA)和甲基丙烯酰化硫酸软骨素(CSMA)。
在本发明一较佳的实施方式中，所述仿生组织支架为骨支架，所述可凝胶成分包括海藻酸钠(Alg)、甲基丙烯酰化明胶(GelMA)和羟基磷灰石(HAp)。
在本发明一较佳的实施方式中，所述仿生组织支架为神经导管支架，所述可凝胶成分成分包括海藻酸钠和甲基丙烯酰化明胶。
在本发明一较佳的实施方式中，所述仿生组织支架为皮肤支架，所述可凝胶成分成分包括甲基丙烯酰化胶原和甲基丙烯酰化明胶。
在本发明一较佳的实施方式中，所述仿生组织支架为肌肉支架，所述可凝胶成分成分包括甲基丙烯酰化透明质酸和甲基丙烯酰化明胶。
本发明中，所述凝胶介质可为本领域常规，较佳地为纯化水、生理盐水、细胞培养基、钙盐溶液和磷酸盐缓冲溶液(PBS溶液)中的一种或多种。其中，所述生理盐水即为0.9％NaCl水溶液。所述细胞培养基可为本领域常规的细胞培养基，例如DMEM、DMEM/F12、RPMI 1640等常用培养基。所述磷酸盐缓冲溶液可为本领域常规，所述磷酸盐缓冲溶液的pH较佳地为7.4。
本发明中，所述甲基丙烯酰化明胶可为本领域常规，可商购，也可采用本领域常规的方法对明胶(Gel)进行甲基丙烯酰化得到。
所述甲基丙烯酰化明胶的甲基丙烯酰化程度可为30％～100％，较佳地为40％～80％。其中，所述甲基丙烯酰化明胶的甲基丙烯酰化程度利用核磁共振氢谱( 1H NMR)来计算，具体为：选取苯丙氨酸标准峰(7.1～7.4ppm)的积分面积作为1，计算明胶改性前后赖氨酸信号在2.8～2.95ppm处的峰面积下降的百分比，即：
GelMA的甲基丙烯酰化程度＝(Gel的赖氨酸信号在2.8～2.95ppm处的峰面积-GelMA的赖氨酸信号在2.8～2.95ppm处的峰面积)/Gel的赖氨酸信号在2.8～2.95ppm处的峰面积*100％。
所述甲基丙烯酰化透明质酸可为本领域常规，可商购，也可采用本领域常规的方法对透明质酸(HA)进行甲基丙烯酰化得到。所述甲基丙烯酰化透明质酸的分子量可为1～2000kDa，较佳地为100～1000kDa，更佳地为500～950kDa，更佳地为890～950kDa。
所述甲基丙烯酰化透明质酸的甲基丙烯酰化程度可为20％～60％，较佳地为30％～50％。其中，所述甲基丙烯酰化透明质酸的甲基丙烯酰化程度利用核磁共振氢谱(1H NMR)来计算，具体为：
HAMA的甲基丙烯酰化程度＝甲基丙烯酰胺-乙烯基在5.6ppm处的峰面积/N-乙酰基葡萄糖在1.9ppm处的峰面积*100％。
所述甲基丙烯酰化硫酸软骨素可为本领域常规，可商购，也可采用本领域常规的方法对硫酸软骨素(CS)进行甲基丙烯酰化得到。所述甲基丙烯酰化硫酸软骨素的分子量可为10～70kDa，较佳地为30～50kDa。
所述甲基丙烯酰化硫酸软骨素的甲基丙烯酰化程度可为20％～60％，较佳地为30％～50％。其中，所述甲基丙烯酰化硫酸软骨素的甲基丙烯酰化程度利用核磁共振氢谱( 1H NMR)来计算，具体为：
CSMA的甲基丙烯酰化程度＝甲基丙烯酰胺-乙烯基在5.6ppm处的峰面积/N-乙酰基葡萄糖在1.9ppm处的峰面积*100％。
本发明中，当所述可凝胶成分为光敏性可凝胶成分时，所述水凝胶组合物还包括光 引发剂。当所述水凝胶组合物包含光引发剂时，在使用时，先将所述光敏性可凝胶成分与凝胶介质混合，再加入所述光引发剂。这样做的目的是，先将所述可凝胶成分溶解于所述凝胶介质中，以便于水凝胶组合物的稳定保存，在使用时临时加入所述光引发剂，避免保存过程中光引发剂的存在导致水凝胶组合物一定程度的交联。
本发明中，所述光引发剂可为本领域常规的光引发剂，较佳地为蓝光引发剂、紫外光引发剂或绿光引发剂；所述蓝光引发剂较佳地为苯基-2,4,6-三甲基苯甲酰基膦酸锂(LAP)、核黄素、黄素单核苷酸、曙红Y或三联吡啶氯化钌/过硫酸钠(Ru/SPS)；所述紫外光引发剂较佳地为2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮(I2959)。
其中，所述水凝胶组合物还可包括增稠剂。所述增稠剂可为本领域常规，较佳地为聚环氧乙烷(PEO)、聚乙二醇(PEG)、海藻酸钠(Alg)、透明质酸、聚乙烯吡咯烷酮、阿拉伯树胶、结冷胶和黄原胶中的一种或多种。
其中，所述水凝胶组合物还可包括合成类光敏材料。所述合成类光敏材料可为本领域常规，较佳地包括聚乙二醇丙烯酸酯(PEGDA)、聚丙烯酸、聚甲基丙烯酸、聚丙烯酸酯、聚甲基丙烯酸酯、聚丙烯酰胺和聚甲基丙烯酰胺中的一种或多种。所述合成类光敏材料较佳地为聚乙二醇丙烯酸酯。
本发明中，所述交联固化的方式可包括物理交联固化、化学交联固化和光交联固化中的一种或多种；较佳地包括光交联固化。所述交联固化可根据所述可凝胶成分的特性选用本领域常规的方法进行。
其中，所述物理交联固化可采用本领域常规的方法进行，例如胶原蛋白在37℃左右的自组装固化。
其中，所述化学交联固化可采用本领域常规的方法进行，例如，甲基丙烯酰化材料在过硫酸铵催化下形成凝胶体，或者，海藻酸钠与二价金属阳离子交联形成凝胶体。
其中，所述光交联固化可采用本领域常规的方法在光照射下进行；较佳地，所述光交联在波长365～405nm、强度5～50mW/cm 2的光照射下进行；更佳地，所述光交联在波长405nm、强度10mW/cm 2的光照射下进行。当所述可凝胶成分为光敏性可凝胶成分时，所述交联固化为光交联固化。
步骤S3中，所述溶剂可根据形成模具的牺牲材料的特性进行选择，可溶解所述模具即可。所述溶剂较佳地为二氯甲烷、三氯甲烷、四氢呋喃、1,4-二氧六环、纯化水、生理盐水、钙盐溶液、磷酸盐缓冲溶液(PBS)或培养基。
步骤S4中，所述冷冻干燥的时间较佳地为8～24h；所述冷冻干燥前较佳地进行预冷步骤；所述预冷的温度较佳地为-20℃，所述预冷的时间较佳地为1～3h。
本发明还提供一种仿生组织支架，其根据所述的仿生组织支架的制备方法制得。
本发明中，所述仿生组织支架可仿生本领域常规的需要仿生的组织，例如软骨、骨、神经导管、韧带、肌肉、乳房、脂肪、皮肤、心脏、肝脏、脾脏、肺脏、肾脏、胰腺、胃、肠、膀胱、血管等。
本发明中所述仿生组织支架的制备方法为间接3D打印法，又称为3D打印脱模法或3D工程法，通过选择合适的牺牲材料，可实现水凝胶材料的高精度3D工程制造，获得的仿生组织支架具有微观精细网格状结构(贯通结构)，孔隙连通，孔隙率可调节(可高达30％～70％)，比表面积大(以形成边长10mm，高度为3mm大小的支架为例，3D打印通孔结构形成的表面积(填充率为50％，层高为0.2mm喷嘴大小为0.25mm)为3200mm 2，而灌注形成同样尺寸的支架，表面积为320mm 2，有孔支架结构较无孔结构表面积增大10倍)。
本发明特别提供一种骨软骨支架，其包括软骨层、黏连层和骨层，所述黏连层的两侧分别与所述软骨层和所述骨层连接；所述软骨层、所述黏连层和所述骨层中的一种或多种为多孔结构。
本发明中，较佳地，所述软骨层、所述黏连层和所述骨层均为多孔结构；更佳地，所述软骨层的孔、所述黏连层的孔与所述骨层的孔连通。所述软骨层的孔、所述黏连层的孔与所述骨层的孔可完全对齐或不完全对齐，较佳地为完全对齐。
本发明中，所述软骨层和/或所述骨层的孔的孔径为50～350μm，较佳地为200～280μm，例如250μm；较佳地，所述骨层的孔的孔径与所述软骨层的孔的孔径相等。所述软骨层和所述骨层的孔径的选择均是为了适合捕获细胞和细胞生长。
本发明中，所述软骨层和/或所述骨层的孔的分布方式较佳地为垂直交叉排列。
本发明中，所述软骨层和/或所述骨层的孔隙率为20％～70％，较佳地为40％～60％，例如50％。
本发明中，所述骨软骨支架的孔隙率可为20％～70％，较佳地为40％～60％，例如50％。
本发明中，所述黏连层是指所述软骨层和所述骨层之间的过渡层，能够实现所述软骨层和所述骨层的连接，不一定通过粘结作用连接。
较佳地，所述黏连层不覆盖或部分覆盖所述骨层和/或所述软骨层的孔。也就是说，所述黏连层仅覆盖所述骨层和所述软骨层的非孔区域的部分或全部，以保证黏连层不堵塞骨层和软骨层的孔。所述黏连层的孔可与所述软骨层的孔和所述骨层的孔一致，保证三层贯通。
本发明中，所述骨软骨支架的形状不做特别限制，在使用中，可根据缺损部位大小 尺寸对所述骨软骨支架进行裁剪即可。
例如，所述骨软骨支架为圆柱体。所述圆柱体的直径可为2～30mm，较佳地为2～20mm，更佳地为3～10mm；所述圆柱体的高度可为2～10mm，较佳地为3～6mm。
例如，所述骨软骨支架为长方体。所述长方体的底面可为正方形，所述正方形的边长可为2～30mm，较佳地为2～20mm，更佳地为3～10mm；所述长方体的高度较佳地为2～10mm，更佳地为3～6mm。
本发明中，所述骨层和所述软骨层的高度比可为1：(0.1～1)，较佳地为1：(0.2～0.5)。
本发明中，所述黏连层的高度可为5μm～2mm，较佳地为0.1～2mm，更佳地为0.5～1mm。
本发明中，所述软骨层的材料可为本领域常规的软骨层材料，较佳地为水凝胶材料。其中，所述水凝胶材料可为单网络水凝胶材料、互穿网络水凝胶材料和复合交联水凝胶材料中的一种或多种。由单一交联方式形成的水凝胶材料，称为单网络水凝胶材料。由两种或两种以上交联方式形成的水凝胶材料，称为互穿网络水凝胶材料，或双网络水凝胶材料。由同一交联方式的多种可凝胶成分复合交联而成，称为复合交联水凝胶材料。所述水凝胶材料较佳地为光交联水凝胶材料，更佳地为复合光交联水凝胶材料。
本发明中，所述软骨层较佳地还负载软骨促进成分。其中，所述软骨促进成分可包括生物活性因子和/或细胞。其中，所述生物活性因子较佳地包括转化生长因子TGFα或TGFβ。所述细胞可包括自体或异体软骨细胞、间充质干细胞、胚胎干细胞或诱导性多能干细胞。
本发明中，所述骨层的材料可为本领域常规的医用高分子材料，较佳地为聚乳酸(PLA)、聚乳酸-羟基乙酸共聚物(PLGA)或聚己内酯(PCL)。
本发明中，所述骨层的材料也可为水凝胶材料，所述水凝胶材料如前所述。
本发明中，所述骨层较佳地还负载骨促进成分。其中，所述骨促进成分可包括生物活性的无机材料、生物活性因子和细胞中的一种或多种。
其中，所述生物活性的无机材料较佳地包括羟基磷灰石、磷酸钙、碳酸钙和生物活性玻璃中的一种或多种。所述生物活性的无机材料在所述骨层中的质量百分比可为0.1wt％～70wt％，较佳地为1wt％～50wt％，更佳地为2.5wt％～30wt％。
所述生物活性因子较佳地包括转化生长因子TGFα、TGFβ，骨形态发生蛋白BMP-2、BMP-3、BMP-4、BMP-5、BMP-6、BMP-7、BMP-8和BMP-9，和软骨诱导化合物(如KGN等)中的一种或多种。
所述细胞可包括自体或异体骨细胞、间充质干细胞、胚胎干细胞或诱导性多能干细 胞。
在某些实施方案中，所述黏连层的材料可为水凝胶材料，所述水凝胶材料如前所述。
在某些实施方案中，所述黏连层可由本领域常规的医用胶水形成。所述医用胶水例如可选自康派特、绿海(Greensea)、金象、海氏海诺、双一、吗丁啉、3M、七乐康、福爱乐、爱达宝(IDEALPLAST)、开颜或奥非特。
本发明中，较佳地，所述软骨层、所述黏连层和所述骨层均为水凝胶材料。此时，所述软骨层、所述黏连层和所述骨层中可凝胶成分在浓度上渐变。
本发明还提供一种所述骨软骨支架的制备方法，其包括以下步骤：将骨层和软骨层连接，连接处形成黏连层；所述软骨层、所述黏连层和所述骨层中的一种或多种为多孔结构。
本发明中，所述软骨层的制备方法可为本领域常规，一般为以水凝胶组合物为原料经交联固化制备。其中，所述水凝胶组合物、所述交联固化的方式如前所述。
在本发明一较佳的实施方案中，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶1～50份，甲基丙烯酰化透明质酸0～30份，甲基丙烯酰化硫酸软骨素0～30份，光引发剂0.01～1份和凝胶介质。
其中，所述甲基丙烯酰化明胶的用量较佳地为1～30份，更佳地为1～20份，更佳地为2～15份，更佳地为5～15份，例如8份、10份或12份。
其中，所述甲基丙烯酰化透明质酸的用量较佳地为0.1～20份，更佳地为0.5～10份，更佳地为1～3份，例如1.5份或2份。
其中，所述甲基丙烯酰化硫酸软骨素的用量较佳地为0.1～20份，更佳地为0.5～20份，更佳地为0.5～5份，更佳地为1～3份，例如1份、2份、2.5份或3份。
其中，所述甲基丙烯酰化明胶和所述甲基丙烯酰化透明质酸的质量比可为(1-30)：(0.5～10)，较佳地为(2～15)：(1～3)，例如5:2。
其中，所述甲基丙烯酰化明胶、所述甲基丙烯酰化透明质酸和所述甲基丙烯酰化硫酸软骨素的质量比可为(1～30)：(0.5～10)：(0.5～20)，较佳地为(2～15)：1：(1～3)，例如10:1:3，5:2:2，15:1:1或8:1:3。
其中，所述凝胶介质的用量可为本领域常规，较佳地使得所述水凝胶组合物中：甲基丙烯酰化明胶(GelMA)5％～30％，甲基丙烯酰化透明质酸(HAMA)0.5～2％，甲基丙烯酰化硫酸软骨素(CSMA)0.1％～5％，光引发剂0.01～1％；其中百分比为每100mL凝胶介质含有的组分质量(g)。
其中，所述软骨层的水凝胶组合物还可包括增稠剂，所述增稠剂的用量较佳地为 0.1～25份。所述增稠剂如前所述。
当所述增稠剂包括海藻酸钠时，所述海藻酸钠的用量较佳地为0.5～2份。当所述增稠剂包括透明质酸时，所述透明质酸的用量较佳地为0.5～2份。当所述增稠剂包括聚乙烯吡咯烷酮时，所述聚乙烯吡咯烷酮的用量较佳地为2～10份。当所述增稠剂包括阿拉伯树胶时，所述阿拉伯树胶的用量较佳地为0.1～25份。当所述增稠剂包括结冷胶时，所述结冷胶的用量较佳地为0.1～2份。当所述增稠剂包括黄原胶时，所述黄原胶的用量较佳地为0.1～5份。
其中，所述软骨层的水凝胶组合物还可包括合成类光敏材料。所述合成类光敏材料的用量较佳地为5～30份。所述合成类光敏材料如前所述。
本发明中，较佳地，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)5～15份，甲基丙烯酰化透明质酸(HAMA)0.5～2份，和光引发剂0.1～0.5份。
本发明中，较佳地，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)5～15份，甲基丙烯酰化透明质酸(HAMA)0.5～2份，甲基丙烯酰化硫酸软骨素(CSMA)0.5～3份，和光引发剂0.1～0.5份。
本发明中，较佳地，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)5～15份，海藻酸钠(Alg)1～2份，和光引发剂0.1～0.5份。
在本发明一具体的实施方案中，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)5份，甲基丙烯酰化透明质酸(HAMA)2份，甲基丙烯酰化硫酸软骨素(CSMA)2份，和光引发剂0.25份。
在本发明一具体的实施方案中，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)10份，甲基丙烯酰化透明质酸(HAMA)1份，甲基丙烯酰化硫酸软骨素(CSMA)3份，和光引发剂0.25份。
在本发明一具体的实施方案中，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)15份，甲基丙烯酰化透明质酸(HAMA)1份，甲基丙烯酰化硫酸软骨素(CSMA)1份，和光引发剂0.25份。
在本发明一具体的实施方案中，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)8份，甲基丙烯酰化透明质酸(HAMA)1份，甲基丙烯酰化硫酸软骨素(CSMA)3份，和光引发剂0.25份。
在本发明一具体的实施方案中，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)8份，甲基丙烯酰化透明质酸(HAMA)1份，甲 基丙烯酰化硫酸软骨素(CSMA)3份，海藻酸钠2份，和光引发剂0.25份。
在本发明一具体的实施方案中，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)8份，甲基丙烯酰化透明质酸(HAMA)1份，甲基丙烯酰化硫酸软骨素(CSMA)3份，PEGDA 10份，和光引发剂1份。
在本发明一具体的实施方式中，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)5份，甲基丙烯酰化透明质酸(HAMA)2份，和光引发剂0.5份。
在本发明一具体的实施方式中，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)5份，海藻酸钠2份，和光引发剂0.5份。
本发明中，所述软骨层的制备方法可为灌注法，其包括以下步骤：将所述软骨层的水凝胶组合物灌注于软骨模具中进行光交联，得到固化水凝胶；将所述固化水凝胶冷冻干燥即得。
其中，所述软骨模具可采用本领域常规的方法根据所需软骨的形状和大小设计而成。
本发明中，所述软骨层的制备方法可为直接3D打印法，其包括以下步骤：将所述软骨层的水凝胶组合物进行3D打印同时进行光交联，得固化水凝胶；将所述固化水凝胶冷冻干燥即得。
在一较佳的实施方式中，所述软骨层的制备方法包括：将水凝胶组合物采用挤出式3D打印机打印，蓝光固化成型，其中，保温温度为30～37℃，打印环境温度为22～25℃，打印压力为20～40PSI，打印速度为4～8mm/s，填充率为40％～60％，光照强度为5～20mW/cm 2。
本发明中，所述软骨层的制备方法较佳地为如前所述的仿生组织支架的制备方法，即3D打印脱模法，具体包括以下步骤：
S1、将牺牲材料进行3D打印，得到软骨层模具；其中，所述牺牲材料为可溶于溶剂的硬质高分子材料；
S2、将软骨层的水凝胶组合物灌注于所述软骨层模具中进行交联，得到固化水凝胶-软骨层模具复合体；
S3、脱模：用溶剂溶解所述软骨层模具，得到固化水凝胶；
S4、将所得固化水凝胶冷冻干燥即得所述软骨层。
在上述实施方案中，所述牺牲材料、所述硬质高分子材料、所述3D打印的方式、所述交联的操作和条件、所述溶剂、所述冷冻干燥均如前所述。
本发明中，所述软骨层的制备方法较佳地还包括负载软骨促进成分的步骤。所述负 载软骨促进成分的方法可为本领域常规。所述软骨促进成分如前所述。当所述软骨促进成分为生物活性因子时，所述生物活性因子的负载方法一般为将所述软骨层在生物活性因子溶液中浸泡，其中，所述生物活性因子溶液的浓度可为1μg/mL～200μg/mL，较佳地为5μg/mL～100μg/mL。
本发明中，所述骨层的制备方法可为本领域常规，较佳地为对所述骨层的材料进行3D打印。其中，所述骨层的材料如前所述。所述3D打印的参数可根据所述骨层的结构和材料采用本领域常规的方法进行选择。所述3D打印较佳地采用熔融沉积式3D打印机进行。
本发明中，较佳地，所述骨层的制备方法还包括负载骨促进成分的步骤。所述负载骨促进成分的方法可为本领域常规，例如：将所述骨层的材料磨成粉末后掺入所述骨促进成分；或者，将所述骨层的材料溶于溶剂后掺入所述骨促进成分，然后使所述溶剂挥发即可。所述骨促进成分如前所述。所述溶剂可为有机溶剂。当所述骨促进成分为生物活性因子时，所述生物活性因子的负载方法一般为将所述骨层在生物活性因子溶液中浸泡，其中，所述生物活性因子溶液的浓度可为1μg/mL～200μg/mL，较佳地为5μg/mL～100μg/mL。
在一较佳的实施方式中，所述骨层通过采用熔融沉积式3D打印机对负载羟基磷灰石的PLA进行3D打印来提供，其中，打印头温度为210℃，平台温度为50～60℃，打印速度为50～60mm/s，填充率为40％～60％。
在一较佳的实施方式中，所述骨层通过采用熔融沉积式3D打印机对负载有磷酸三钙的PCL进行3D打印来提供，其中，打印温度为140～150℃，打印速度为50～60mm/s，填充率为40％～60％。
在一较佳的实施方式中，所述骨层通过采用挤出式光固化3D打印机对负载有磷酸三钙的GelMA进行3D打印来提供，其中，保温温度为30～35℃，打印环境温度为22～25℃，打印压力为20～40PSI，打印速度为4-8mm/s，填充率为40-60％，光照强度为5～50mW/cm 2。
本发明中，所述骨层的制备方法也可为以水凝胶组合物为原料经交联固化制备。当所述骨层以水凝胶组合物为原料经交联固化制备时，所述水凝胶组合物、所述交联固化的方式如前所述。
在一较佳的实施方式中，所述骨层的水凝胶组合物包括以下组分：甲基丙烯酰化明胶(GelMA)5％～30％，生物活性玻璃2.5％～50％和光引发剂0.01％～1％；其中百分比为每100mL凝胶介质含有的组分质量(g)。
本发明中，所述连接一般地为通过医用胶水将所述骨层和所述软骨连接。所述连接的操作条件可根据所用医用胶水的使用方法确定即可。所述医用胶水如前所述。
在某些实施方案中，所述骨软骨支架采用如前所述的仿生组织支架的制备方法，即3D打印脱模法，进行骨软骨一体成型，具体包括以下步骤：
S1、将牺牲材料进行3D打印，得到骨软骨支架模具；其中，所述牺牲材料为可溶于溶剂的硬质高分子材料；
S2、依次将骨层、黏连层和软骨层的水凝胶组合物灌注于所述骨软骨支架模具中进行交联，得到固化水凝胶-骨软骨支架模具复合体；
S3、脱模：用溶剂溶解所述骨软骨支架模具，得到固化水凝胶；
S4、将所得固化水凝胶冷冻干燥即得所述骨软骨支架。
在上述实施方案中，较佳地，所述骨层的水凝胶组合物包括以下组分：甲基丙烯酰化明胶(GelMA)5％～30％，生物活性玻璃2.5％～50％和光引发剂0.01％～1％；所述黏连层的水凝胶组合物包括以下组分：甲基丙烯酰化明胶(GelMA)5％～30％，生物活性玻璃10％～20％和光引发剂0.01％～1％；所述软骨层的水凝胶组合物包括以下组分：甲基丙烯酰化明胶(GelMA)5％～30％，甲基丙烯酰化透明质酸(HAMA)0.5％～2％，甲基丙烯酰化硫酸软骨素(CSMA)0.5％～5％，和光引发剂0.01％～1％；其中，百分比为每100mL凝胶介质含有的组分质量(g)。
在上述实施方案中，所述牺牲材料、所述硬质高分子材料、所述3D打印的方式、所述交联的操作和条件、所述溶剂、所述冷冻干燥均如前所述。
本发明还提供一种所述骨软骨支架在修复软骨缺损中的应用。
本发明中，所述软骨缺损可为骨软骨复合缺损或单纯软骨缺损。所述骨软骨缺损可位于膝关节、髋关节或肩关节。
在修复骨软骨复合缺损中，所述骨软骨支架的使用方式可为：在骨软骨复合缺损处向下钻孔至骨层，达到骨髓腔后，去除多余的骨层基质，将骨软骨支架材料放入骨软骨复合缺损处。此时，骨层流出的骨髓中富含大量的间充质干细胞，在流经所述骨软骨支架材料时，骨髓源间充质干细胞被捕获于支架中。手术完成后，缝合伤口，即完成了骨软骨复合缺损的修复。
在修复单纯软骨缺损中，所述骨软骨支架的使用方式可为：将所述骨软骨支架的软骨层放置于缺损处，在软骨缺损处进行微骨折处理，至骨髓流出。此时，骨层冒出的骨髓中富含大量的间充质干细胞，在流经所述骨软骨支架时，细胞被捕获于支架中。手术完成后，缝合伤口，即完成了单纯软骨缺损的修复。
本发明的骨软骨支架具有以下优势：(1)本发明的骨软骨支架具有精细化的通孔结构，保证上下、水平的贯通，有利于支架在填充骨软骨缺损时充分地捕获细胞，还有利于营养物质和代谢废物的运输，从而有利于缺损修复。进一步地，通过选用合适的软骨层材料和骨层材料可以诱导干细胞分化生长成为软骨细胞和骨细胞。(2)本发明的骨软骨支架具有优异的黏连层结构，能够牢固连接软骨层和骨层，达到一体化整合的目的；黏连层作为骨层的过渡层，防止软骨层骨化。本发明的骨软骨支架可以实现骨软骨全层修复。(3)本发明的骨软骨支架具备简便的临床操作方式和实用性，为临床上软骨缺损及骨软骨复合缺损的修复提供了一种全新的、有效的解决方案。
本发明还提供一种用于仿生软骨支架的水凝胶组合物，其包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)1～50份，甲基丙烯酰化透明质酸(HAMA)0.1～30份，甲基丙烯酰化硫酸软骨素(CSMA)0.1～30份，光引发剂0.01～1份和凝胶介质。
本发明中，所述甲基丙烯酰化明胶的用量较佳地为1～20份，更佳地为5～15份，例如8份、10份或12份。
本发明中，所述甲基丙烯酰化明胶的甲基丙烯酰化程度可为30％～100％，较佳地为40％～80％。
本发明中，所述甲基丙烯酰化透明质酸的用量较佳地为0.1～10份，更佳地为0.5～10份，进一步更佳地为0.5～2份，例如0.5份、1份、1.5份。
本发明中，所述甲基丙烯酰化透明质酸的分子量可为1～8000kDa，较佳地为100～1000kDa，更佳地为500～950kDa。
本发明中，所述甲基丙烯酰化透明质酸的甲基丙烯酰化程度可为20％～60％，较佳地为30％～50％。
本发明中，所述甲基丙烯酰化硫酸软骨素的用量较佳地为0.1～10份，更佳地为0.5～10份，进一步更佳地为0.5～3份，例如1份、2份或2.5份。
本发明中，所述甲基丙烯酰化硫酸软骨素的分子量可为5～50kDa，较佳地为10～40kDa。
本发明中，所述甲基丙烯酰化硫酸软骨素的甲基丙烯酰化程度可为20％～60％，较佳地为30％～50％。
本发明中，所述甲基丙烯酰化明胶、所述甲基丙烯酰化透明质酸和所述甲基丙烯酰化硫酸软骨素的质量比较佳地为(2-15)：(0.5-5)：(1-5)，更佳地为(2-15)：1：(1-3)，例如10:1:3，5:2:2，15:1:1或8:1:3。
本发明中，所述光引发剂的种类如前所述。所述光引发剂的用量较佳地为0.1～0.5份， 例如0.25份。
本发明中，较佳地，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)5～15份，甲基丙烯酰化透明质酸(HAMA)0.5～10份，甲基丙烯酰化硫酸软骨素(CSMA)0.5～10份，和光引发剂0.1～0.5份。
在本发明一较佳的实施方案中，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)5份，甲基丙烯酰化透明质酸(HAMA)2份，甲基丙烯酰化硫酸软骨素(CSMA)2份，和光引发剂0.25份。
在本发明一较佳的实施方案中，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)10份，甲基丙烯酰化透明质酸(HAMA)1份，甲基丙烯酰化硫酸软骨素(CSMA)3份，和光引发剂0.25份。
在本发明一较佳的实施方案中，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)15份，甲基丙烯酰化透明质酸(HAMA)1份，甲基丙烯酰化硫酸软骨素(CSMA)1份，和光引发剂0.25份。
在本发明一较佳的实施方案中，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)8份，甲基丙烯酰化透明质酸(HAMA)1份，甲基丙烯酰化硫酸软骨素(CSMA)3份，和光引发剂0.25份。
在本发明一较佳的实施方案中，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)8份，甲基丙烯酰化透明质酸(HAMA)1份，甲基丙烯酰化硫酸软骨素(CSMA)3份，海藻酸钠2份，和光引发剂0.25份。
在本发明一较佳的实施方案中，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶(GelMA)8份，甲基丙烯酰化透明质酸(HAMA)1份，甲基丙烯酰化硫酸软骨素(CSMA)3份，PEGDA 10份，和光引发剂1份。
本发明中，所述用于仿生软骨支架的水凝胶组合物还可包括增稠剂，所述增稠剂的用量较佳地为0.1～25份。所述增稠剂的种类如前所述。
其中，当所述增稠剂包括海藻酸钠时，所述海藻酸钠的用量较佳地为1～2份。当所述增稠剂包括透明质酸时，所述透明质酸的用量较佳地为0.5～2份。当所述增稠剂包括聚乙烯吡咯烷酮时，所述聚乙烯吡咯烷酮的用量较佳地为2～10份。当所述增稠剂包括阿拉伯树胶时，所述阿拉伯树胶的用量较佳地为0.1～25份。当所述增稠剂包括结冷胶时，所述结冷胶的用量较佳地为0.1～2份。当所述增稠剂包括黄原胶时，所述黄原胶的用量较佳地为0.1～1份。
本发明中，所述用于仿生软骨支架的水凝胶组合物还可包括合成类光敏材料。所述 合成类光敏材料的用量较佳地为5～30份。所述合成类光敏材料的种类如前所述。
本发明中，所述凝胶介质如前所述。所述凝胶介质的用量可为本领域常规，较佳地使得所述用于仿生软骨支架的水凝胶组合物中：甲基丙烯酰化明胶(GelMA)5％-20％，甲基丙烯酰化透明质酸(HAMA)0.1％～3％，甲基丙烯酰化硫酸软骨素(CSMA)0.1％～5％，光引发剂0.01％～1％；其中百分比为每100mL凝胶介质含有的组分质量(g)。
本发明还提供一种仿生软骨支架的制备方法，其采用所述用于仿生软骨支架的水凝胶组合物为原料，经光交联固化得到。
本发明中，所述仿生软骨支架的制备方法较佳地为如前所述的仿生组织支架的制备方法，即3D打印脱模法，具体包括以下步骤：
S1、将牺牲材料进行3D打印，得到仿生软骨支架模具；其中，所述牺牲材料为可溶于溶剂的硬质高分子材料；
S2、将软骨层的水凝胶组合物灌注于所述仿生软骨支架模具中进行交联，得到固化水凝胶-仿生软骨支架模具复合体；
S3、脱模：用溶剂溶解所述仿生软骨支架模具，得到固化水凝胶；
S4、将所得固化水凝胶冷冻干燥即得所述仿生软骨支架模具。
在上述实施方案中，所述牺牲材料、所述硬质高分子材料、所述3D打印的方式、所述交联的操作和条件、所述溶剂、所述冷冻干燥均如前所述。
本发明中，所述仿生软骨支架的制备方法可为灌注法，所述灌注法的操作如前所述。
本发明中，所述仿生软骨支架的制备方法可为直接3D打印法，所述直接3D打印法的操作如前所述。
在上述仿生软骨支架的制备方法中，所述光交联固化如前所述。
本发明还提供一种仿生软骨支架，其由所述仿生软骨支架的制备方法制得。
本发明还提供一种所述用于仿生软骨支架的水凝胶组合物或所述仿生软骨支架在骨软骨组织工程中的应用。
本发明提供的用于仿生软骨支架的水凝胶组合物可通过灌注或3D打印等方法制得仿生软骨支架，制备方法简单可行。所制得的仿生软骨支架具有以下优点：(1)生物兼容性好；(2)可降解，被再生软骨取代；(3)抑制骨化成骨；(4)诱导软骨形成；(5)3D打印脱模法可得到高精度通孔仿生软骨支架。
在符合本领域常识的基础上，上述各优选条件可任意组合，即得本发明各较佳实例。
附图说明
图1为本发明实施例1～3的软骨支架的模具设计图。
图2为本发明实施例1的软骨支架的模具的显微镜照片。
图3为本发明实施例1的软骨支架的显微镜照片。
图4为本发明实施例2的软骨支架的模具的相机照片。
图5为本发明实施例2的软骨支架的相机照片。
图6为本发明实施例4的骨支架的模具设计图。
图7为本发明实施例4的骨支架的模具的相机照片。
图8为本发明实施例4的固化水凝胶复水后相机照片。
图9为本发明实施例5的神经导管支架的模具设计图.
图10为本发明实施例5的神经导管支架的模具打印实物图。
图11为本发明实施例5的神经导管支架的固化凝胶-模具复合体图。
图12为本发明实施例5的导管支架脱模后的图片。
图13为本发明实施例6的皮肤支架的模具设计图。
图14为本发明实施例6的皮肤支架的模具打印实物图。
图15为本发明实施例6的皮肤支架的固化凝胶-模具复合体图。
图16为本发明实施例6的皮肤支架脱模后的图片。
图17为本发明实施例7的肌肉支架的模具设计图。
图18为本发明实施例7的肌肉支架的模具打印实物图。
图19为本发明实施例7的肌肉支架的固化凝胶-模具复合体图。
图20为本发明实施例7的肌肉支架脱模后的图片。
图21为MSC细胞在本发明实施例1的软骨支架上培养的显微镜照片。
图22为本发明实施例8～11中骨软骨支架的结构示意图。
图23为本发明实施例8的骨软骨支架的相机照片。
图24为本发明实施例8中骨软骨支架的显微镜照片。
图25为本发明效果实施例2中MSC细胞在骨软骨支架上培养的显微镜照片。
图26为本发明效果实施例3中(a)组损伤关节的大体观察照片。
图27为本发明效果实施例3中(b)组损伤关节的大体观察照片。
图28为本发明效果实施例3中(c)组损伤关节的大体观察照片。
图29为本发明效果实施例4的水凝胶在光照下模量随时间的变化。
图30为本发明效果实施例5的固化水凝胶支架的应力-应变曲线。
图31为细胞在本发明效果实施例6的固化水凝胶中培养7天后的存活率(活细胞绿染，死细胞红染)。
图32为本发明效果实施例7中大体观察照片。
图33为本发明效果实施例7中组织切片图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1
软骨支架的制备方法，其包括以下步骤：
S1、根据所需软骨支架设计模具(如图1所示)，用PLA作为牺牲材料，采用熔融堆积方式打印模具(如图2所示)；
S2、向模具中灌注水凝胶组合物，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联固化，再浸泡0.1M CaCl 2溶液进行化学交联固化，得到固化水凝胶-模具复合体；
其中，所述水凝胶组合物的制备过程如下：称取0.2g海藻酸钠(Alg)，0.5g GelMA和50mg LAP溶于10mL PBS溶液(pH＝7.4)中，在37℃下配制而成；
S3、将固化水凝胶-模具复合体浸泡在二氯甲烷中，二氯甲烷将模具溶解；
S4、将所得的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干8h，即得软骨支架(如图3所示)。
实施例2
软骨支架的制备方法，其包括以下步骤：
S1、根据所需软骨支架设计模具(如图1所示)，用PETG作为牺牲材料，采用熔融堆积方式打印模具(如图4所示)；
S2、向模具中灌注水凝胶组合物，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联固化，得到固化水凝胶-模具复合体；
其中，所述水凝胶组合物的制备过程如下：称取0.5g GelMA，0.2g HAMA和50mg LAP溶于10mL PBS溶液(pH＝7.4)中，在37℃下配制而成；
S3、将固化水凝胶-模具复合体浸泡在二氯甲烷中，二氯甲烷将模具溶解；
S4、将所得的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干20h，即得软骨支架(如图5所示)。
实施例3
软骨支架的制备方法，其包括以下步骤：
S1、根据所需软骨支架设计模具(如图1所示)，用PLA生物基光敏树脂(易生新材料有限公司)作为牺牲材料，采用光固化方式(LCD)打印模具；
S2、向模具中灌注水凝胶组合物，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联固化，得到固化水凝胶-模具复合体；
其中，所述水凝胶组合物的制备过程如下：
称取1.0g GelMA，0.2g HAMA，0.2g CSMA和50mg LAP溶于10mL PBS溶液(pH＝7.4)中，在37℃下配制而成。
S3、将固化水凝胶-模具复合体浸泡在二氯甲烷中，二氯甲烷将模具溶解；
S4、将所得的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干12h；即得软骨支架。
实施例4
骨支架的制备方法，其包括以下步骤：
S1、根据所需软骨支架设计模具(如图6所示)，用PVA作为牺牲材料，采用熔融堆积方式打印模具(如图7所示)；
S2、向模具中灌注水凝胶组合物，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联固化，再浸泡0.1M CaCl 2溶液进行物理交联固化，得到固化水凝胶-模具复合体；
其中，所述水凝胶组合物的制备过程如下：称取0.2g海藻酸钠(Alg)，0.5g GelMA，0.1g羟基磷灰石(HAp)和30mg LAP溶于10mL PBS溶液(pH＝7.4)中，在37℃下配制而成；
S3、将固化水凝胶-模具复合体浸泡在纯化水中，将模具溶解；
S4、将所得的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干20h；即得骨支架。图8所示为复水后的照片。
实施例5
神经导管支架的制备方法，其包括以下步骤：
S1、根据所需神经导管支架设计模具(如图9所示)，用PLA作为牺牲材料，采用熔融堆积方式打印模具(如图10所示)；
S2、向模具中灌注水凝胶组合物，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联固化，再浸泡0.1M CaCl 2溶液进行物理交联固化，得到固化水凝胶-模具复合体(如图11所示)；
其中，所述水凝胶组合物的制备过程如下：称取0.2g海藻酸钠(Alg)，0.5g GelMA，和25mg LAP溶于10mL PBS溶液(pH＝7.4)中，在37℃下配制而成；
S3、将固化水凝胶-模具复合体浸泡在二氯甲烷中，二氯甲烷将模具溶解(如图12所示)；
S4、将所得的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干20h；即得神经导管支架。
实施例6
皮肤支架的制备方法，其包括以下步骤：
S1、根据所需皮肤支架设计模具(如图13所示)，用PCL作为牺牲材料，采用熔融堆积方式打印模具(如图14所示)；
S2、向模具中灌注水凝胶组合物，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联固化，得到固化水凝胶-模具复合体(如图15所示)；
其中，所述水凝胶组合物的制备过程如下：称取0.2g ColⅠMA，1.0g GelMA，和10mg LAP溶于10mL PBS溶液(pH＝7.4)中，在37℃下配制而成；
S3、将固化水凝胶-模具复合体浸泡在二氯甲烷中，二氯甲烷将模具溶解(如图16所示)；
S4、将所得的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干20h；即得皮肤支架。
实施例7
肌肉支架的制备方法，其包括以下步骤：
S1、根据所需肌肉支架设计模具(如图17所示)，用PLA作为牺牲材料，采用熔融堆积方式打印模具(如图18所示)；
S2、向模具中灌注水凝胶组合物，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联固化，得到固化水凝胶-模具复合体(如图19所示)；
其中，所述水凝胶组合物的制备过程如下：称取0.2g HAMA，1.0g GelMA，和10mg LAP溶于10mL PBS溶液(pH＝7.4)中，在37℃下配制而成；
S3、将固化水凝胶-模具复合体浸泡在二氯甲烷中，二氯甲烷将模具溶解(如图20所示)；
S4、将所得的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干20h；即得肌肉支架。
效果实施例1：细胞相容性测试
以实施例1制备的软骨支架为例，将间充质干细胞MSC种植于支架材料上，然后加入培养基，在37℃/5％CO 2条件下培养24h。测试前，先将细胞培养液吸出，并用PBS多次洗涤，接着加入l mL细胞活/死双染试剂(10μM的钙黄绿素和15μM的二聚乙啡啶溶于5mL PBS中)，在37℃下与细胞共同孵育30min，然后利用共聚焦荧光显微镜观察支架材料内部细胞的粘附及存活情况。活细胞呈现钙黄素染色活性，433nm下发射绿色荧光；死细胞被溴化乙啶染色，543nm激发下发射红色荧光。从图21中看出，该类支架材料具有较好的细胞相容性，且能够长入支架材料的通孔结构中。
实施例8
1、软骨层的制备
(1)甲基丙烯酰化明胶(GelMA)的合成：将明胶(1g)溶于10mL PBS(pH＝7.4)中，加热至50℃搅拌至完全溶解，加入0.5mL甲基丙烯酸酐，反应2～3h，反应后用40mL PBS稀释反应液，然后倒入透析袋(mWCO 7000)中，用去离子水透析2-3d，冷冻干燥即可得到甲基丙烯酰酯化明胶(0.9g)。根据核磁共振氢谱( 1H NMR)，选取苯丙氨酸标准峰(7.1～7.4ppm)的积分面积作为1，计算明胶改性前后赖氨酸信号在2.8～2.95ppm处的峰面积下降的百分比，即得甲基丙烯酰化明胶的甲基丙烯酰化程度为65％。
(2)甲基丙烯酰化透明质酸(HAMA)的合成：将透明质酸(1g，900kDa)溶于100mL去离子水，冷却至0-4℃，加入5mL甲基丙烯酸酐，再缓慢滴加5mL 5M NaOH水溶液，反应24h，然后将反应液倒入透析袋(mWCO 7000)中，用去离子水透析2-3d，冷冻干燥即可得到甲基丙烯酰化透明质酸(0.9g)。根据核磁共振氢谱( 1H NMR)，计算HAMA的甲基丙烯酰化程度为40％(HAMA的甲基丙烯酰化程度＝甲基丙烯酰胺-乙烯基在5.6ppm处的峰面积/N-乙酰基葡萄糖在1.9ppm处的峰面积*100％)。
(3)配制5％GelMA/2％HAMA/0.5％LAP的水凝胶组合物：称取0.05g GelMA，0.02g HAMA和5mg LAP溶于1mL PBS溶液(pH＝7.4)中，在37℃下配制5％GelMA/2％HAMA/0.5％LAP。
(4)灌注法制备GelMA/HAMA软骨层：
将水凝胶组合物灌注于预制的圆柱体模具(直径5mm，高度3mm)中，在405nm波长、强度10mW/cm 2的光源照射下实现光交联，即可获得GelMA/HAMA光交联固化水凝胶；将制备的GelMA/HAMA光交联固化水凝胶放置于-20℃冰箱中冷冻2h，再用冻干机冻干，即得GelMA/HAMA软骨层。
(5)TGFβ的负载：将上述GelMA/HAMA软骨层浸泡于10μg/mL TGFβ溶液中， 待充分吸附12h后，放置于-20℃冰箱中冷冻2h，再用冻干机冻干，即可获得负载TGFβ的软骨层，记为TGFβ-GelMA/HAMA软骨层。
2、骨层的制备
(1)负载羟基磷灰石的聚乳酸高分子材料(HAP/PLA)的制备
称取5g聚乳酸(PLA)溶于20mL二氯甲烷后至完全溶解，加入1g羟基磷灰石(HAP)，搅拌至均匀溶液，然后置于通风橱待二氯甲烷挥发后，放入真空干燥箱烘干24h，用粉碎机粉碎制成粉末，经拉丝机制成1.75mm线材，即可制成为HAP/PLA高分子材料。
(2)3D打印HAP/PLA高分子材料
将HAP/PLA高分子材料采用熔融沉积式3D打印机打印成型(打印温度：210℃；平台温度为50℃；打印速度：60mm/s；填充率：50％，层高为0.1mm)，得到HAP/PLA骨层。所得HAP/PLA骨层为圆柱体(直径5mm，高度为3mm)，孔隙率50％，孔径为250μm。
3、骨软骨支架的制备
用医用胶水金象将上述TGFβ-GelMA/HAMA软骨层和HAP/PLA骨层连接，连接处形成黏连层，黏连层的厚度约为100μm，即得TGFβ-GelMA/HAMA-HAP/PLA骨软骨支架，其结构示意图见图22，从上至下一次为软骨层1、黏连层2和骨层3；实物相机照片见图23；通过显微镜观察其微观结构，如图24所示。
本实施例中，如果不进行TGFβ的负载，即得GelMA/HAMA-HAP/PLA骨软骨支架。
实施例9
1、软骨层的制备
(1)甲基丙烯酰化明胶(GelMA)的合成：同实施例1。
(2)甲基丙烯酰化透明质酸(HAMA)的合成：同实施例1。
(3)配制5％GelMA/2％HAMA/0.5％LAP水凝胶组合物：同实施例1。
(4)直接3D打印法制备软骨层：
将上述水凝胶组合物采用挤出式3D打印机打印，蓝光固化成型(保温温度：37℃；平台温度：22℃；打印压力：20PSI；打印速度：5mm/s；填充率：50％)；将制备的GelMA/HAMA光交联固化水凝胶放置于-20℃冰箱中冷冻2h，再用冻干机冻干，即得GelMA/HAMA软骨层。所得GelMA/HAMA软骨层为圆柱体(直径为5mm，高度为1mm)，孔隙率为50％，孔径为300μm。
(5)TGFβ的负载：同实施例1。
2、骨层的制备：同实施例1。
3、骨软骨支架的制备
用医用胶水金象将上述TGFβ-GelMA/HAMA软骨层和HAP/PLA骨层连接，连接处形成黏连层，黏连层的厚度约为100μm，即得TGFβ-GelMA/HAMA-HAP/PLA骨软骨支架，其结构示意图见图22。
本实施例中，如果不进行TGFβ的负载，即得GelMA/HAMA-HAP/PLA骨软骨支架。
实施例10
1、软骨层的制备
(1)甲基丙烯酰化明胶(GelMA)的合成：同实施例1。
(2)海藻酸钠/明胶复合交联水凝胶组合物的制备：称取0.02g海藻酸钠(Alg)，0.05g GelMA和5mg LAP溶于1mL PBS溶液(pH＝7.4)中，在37℃下配成2％Alg/5％GelMA/0.5％LAP的水凝胶组合物。
(3)将水凝胶组合物灌注于预制的模具中，在405nm波长、强度10mW/cm 2的光源照射下实现光交联；将成型的水凝胶从模具中取出，于0.1M CaCl 2中浸泡2h，实现化学交联，即可获得复合光交联固化水凝胶；然后，将制备的复合光交联固化水凝胶放置于-20℃冰箱中冷冻2h，再用冻干机冻干，即得Alg/GelMA软骨层。
(4)间充质干细胞(MSC)的负载：将MSC用胰酶消化，离心收集细胞，然后将细胞悬液滴加于上述Alg/GelMA软骨层上，孵育1h后，加入培养基，在37℃/5％CO 2条件的细胞培养箱中培养7d，即可获得MSC-Alg/GelMA软骨层。
2、骨层的制备
(1)负载磷酸三钙(TCP)的聚己内酯高分子材料(TCP/PCL)的制备
称取5g聚己内酯(PCL)溶于20mL二氯甲烷后至完全溶解，加入1g磷酸三钙(TCP)，搅拌至均匀溶液，然后置于通风橱待二氯甲烷挥发后，放入真空干燥箱烘干24h，用粉碎机粉碎制成粉末，即可制成为TCP/PCL高分子材料。
(2)3D打印TCP/PCL高分子材料：将TCP/PCL高分子材料采用熔融沉积式3D打印机打印成型(打印温度：140℃；打印压力：40PSI；打印速度：50mm/s；填充率：50％)，得到TCP/PCL骨层。所得TCP/PCL骨层为圆柱体(直径5mm，高度为3mm)，孔隙率50％，孔径为300μm。
3、骨软骨支架的制备
用医用胶水金象将上述MSC-Alg/GelMA软骨层和TCP/PCL骨层连接，连接处形成黏连层，黏连层的厚度约为100μm，即得MSC-Alg/GelMA-TCP/PCL骨软骨支架，其结 构示意图见图22。
本实施例中，如果不进行MSC的负载，即得Alg/GelMA-TCP/PCL骨软骨支架。
实施例11
1、软骨层的制备
(1)甲基丙烯酰化明胶(GelMA)的合成：同实施例1。
(2)海藻酸钠/明胶复合交联水凝胶组合物的配制：同实施例3。
(3)3D打印脱模法制备Alg/GelMA软骨层：
S1、根据软骨层设计合适的软骨层模具，并将聚丙烯酸酯类光敏树脂进行3D打印成型；
S2、将水凝胶组合物灌注于软骨层模具中，在405nm波长、强度10mW/cm 2的光源照射下实现原位光交联，再于0.1M CaCl 2中浸泡2h，实现化学交联，得到Alg/GelMA复合光交联固化水凝胶-软骨层模具复合体；
S3、脱模：用二氯甲烷溶解Alg/GelMA复合光交联固化水凝胶-软骨层模具复合体中的软骨层模具，即可获得Alg/GelMA复合光交联固化水凝胶；
S4、将制备的Alg/GelMA复合光交联固化水凝胶放置于-20℃冰箱中冷冻2h，再用冻干机冻干，即得Alg/GelMA软骨层。其中，所得Alg/GelMA软骨层为圆柱体(直径为5mm，高度为1mm)，孔隙率为50％，孔径为250μm。
(4)间充质干细胞(MSC)的负载：同实施例3。
2、骨层的制备：同实施例3。
3、骨软骨支架的制备
用医用胶水金象将上述MSC-Alg/GelMA软骨层和TCP/PCL骨层连接，连接处形成黏连层，黏连层的厚度约为100μm，即得MSC-Alg/GelMA-TCP/PCL骨软骨支架，其结构示意图见图22。
本实施例中，如果不进行MSC的负载，即得Alg/GelMA-TCP/PCL骨软骨支架。
实施例12
1、可凝胶成分的合成
(1)甲基丙烯酰化明胶(GelMA)的合成：同实施例1。
(2)甲基丙烯酰化透明质酸(HAMA)的合成：同实施例1。
(3)甲基丙烯酰化硫酸软骨素(CSMA)的合成：将硫酸软骨素(10g，30kDa)溶于100mL去离子水，冷却至0-4℃，加入50mL甲基丙烯酸酐，再缓慢滴加50mL 5M NaOH水溶液，反应24h，然后将反应液倒入透析袋(mWCO 7000)中，用去离子水透 析2～3d，冷冻干燥即可得到甲基丙烯酰化硫酸软骨素(9g)。根据核磁共振氢谱( 1H NMR)，计算CSMA的甲基丙烯酰化程度为40％(CSMA的甲基丙烯酰化程度＝甲基丙烯酰胺-乙烯基在5.6ppm处的峰面积/N-乙酰基葡萄糖在1.9ppm处的峰面积*100％)。
2、配制水凝胶组合物
(1)软骨层的水凝胶组合物：称取20g的甲基丙烯酰化明胶(GelMA)、1g的甲基丙烯酰化透明质酸(HAMA)、1g的甲基丙烯酰化硫酸软骨素(CSMA)，在50℃下溶解于去离子水中，加入0.1g的引发剂LAP，配制成软骨层的水凝胶，其中百分比为每100mL凝胶介质含有的组分质量(g)；
(2)过渡层的水凝胶组合物：称取20g的甲基丙烯酰化明胶(GelMA)、10g的生物活性玻璃，在50℃下溶解于去离子水中，加入0.25g的引发剂LAP，配制成过渡层的水凝胶；其中百分比为每100mL凝胶介质含有的组分质量(g)；
(3)骨层的水凝胶组合物：称取20g的甲基丙烯酰化明胶(GelMA)、50g的生物活性玻璃，在50℃下溶解于去离子水中，加入0.2g的引发剂LAP，配制成骨层的水凝胶；其中百分比为每100mL凝胶介质含有的组分质量(g)。
3、3D打印脱模法一体成型
S1、根据骨软骨支架设计合适的骨软骨支架模具，并将聚乙烯醇(PVA)3D打印成型；
S2、依次将骨层、黏连层和软骨层的水凝胶组合物灌注于骨软骨支架模具中，在405nm波长、强度10mW/cm 2的光源照射下实现原位光交联，得到光交联固化水凝胶-骨软骨支架模具复合体；
S3、脱模：用纯化水溶解光交联固化水凝胶-骨软骨支架模具复合体中的骨软骨支架模具，即可获得光交联固化水凝胶；
S4、将制备的光交联固化水凝胶放置于-20℃冰箱中冷冻2h，再用冻干机冻干，即得一体成型的骨软骨支架。
所得骨软骨支架为长方体(底面为30mm*30mm，高度为3mm)，孔隙率为50％，孔径为250μm。
效果实施例2：骨软骨支架的细胞相容性测试
以实施例8制备的GelMA/HAMA-HAP/PLA骨软骨支架、实施例11制备的Alg/GelMA-TCP/PCL骨软骨支架为例。
将间充质干细胞(MSC)用胰酶消化，离心收集细胞，然后将细胞悬液滴加于上述骨软骨支架上，孵育1h后，加入培养基，在37℃/5％CO 2条件的细胞培养箱中培养24 h。测试前，先将细胞培养液吸出，并用PBS多次洗涤，接着加入lmL细胞活/死双染试剂(10μM的钙黄绿素和15μM的二聚乙啡啶溶于5mL PBS中)，在37℃下与细胞共同孵育30min。
利用共聚焦荧光显微镜观察骨软骨支架内部细胞的粘附及存活情况。活细胞呈现钙黄素染色活性，433nm下发射绿色荧光；死细胞被溴化乙啶染色，543nm激发下发射红色荧光。从图25中看出，本发明的骨软骨支架具有较好的细胞相容性，且能够长入支架材料的通孔结构中。
效果实施例3：骨软骨支架在兔子骨软骨复合缺损修复中的应用
以实施例8制备的GelMA/HAMA-HAP/PLA骨软骨支架为例。
采用新西兰雄性大白兔，每只兔子均建立骨软骨复合缺损模型。实验前按体重随机分组(每组3只)：a：空白对照组；b：骨层支架(HAP/PLA)阴性对照组；c：骨软骨支架(GelMA/HAMA-HAP/PLA)组。在手术中，将支架填充于兔子关节骨软骨复合缺损处。在手术12周后，通过静脉注射空气的方法处死实验中的兔子，并提取损伤关节对实验修复效果进行评价。损伤关节的大体观察照片如图26～28所示。图26为空白对照组，由于没有放置支架，几乎看不到新生组织。图27为仅有骨层支架的阴性对照组，因为软骨层缺失，完全没有长出新生软骨，仅看到尚未降解的骨层支架。图28为骨软骨支架组，可以看出植入骨软骨支架处形成了新的组织，且与周围正常组织有类似的外观，有较好的修复效果。
以下实施例13～18和对比例1～3中，甲基丙烯酰化明胶(GelMA：SR-3DP-0201)、甲基丙烯酰化透明质酸(HAMA：SR-3DP-0301)、甲基丙烯酰化硫酸软骨素(CSMA：SR-3DP-0401)和LAP均购自华夏司印(上海)生物技术有限公司。其中，GelMA的甲基丙烯酰化程度65％；HAMA的分子量为900kDa，甲基丙烯酰化程度40％；CSMA的分子量为30kDa，甲基丙烯酰化程度40％。
实施例13
称取0.05g GelMA、0.02g HAMA、0.02g CSMA和2.5mg LAP，在37℃下溶于1mL 0.9％NaCl溶液(pH＝7.4)中，将该混合物灌注于预制的模具中，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联，即可获得5％GelMA/2％HAMA/2％CSMA固化水凝胶。然后，将制备的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干24h，即可制备5％GelMA/2％HAMA/2％CSMA的仿生软骨支架。
实施例14
称取0.1g GelMA、0.01g HAMA、0.03g CSMA和2.5mg LAP，在37℃下溶于1 mL PBS溶液(pH＝7.4)中，将该混合物灌注于预制的模具中，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联，即可获得10％GelMA/1％HAMA/3％CSMA固化水凝胶。然后，将制备的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干20h，即可制备10％GelMA/1％HAMA/3％CSMA的仿生软骨支架。
实施例15
称取0.15g GelMA、0.01g HAMA、0.01g CSMA和2.5mg LAP，在37℃下溶于1mL PBS溶液(pH＝7.4)中，将该混合物置于蓝光辅助挤出式3D打印机中，在405nm波长、强度10mW/cm 2的蓝光照射下3D打印，即可获得15％GelMA/1％HAMA/1％CSMA固化水凝胶。然后，将制备的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干12h，即可制备15％GelMA/1％HAMA/1％CSMA的仿生软骨支架。
实施例16
33D打印脱模法制备仿生软骨支架：
(1)根据所需仿生软骨支架设计合适的模具，采用PLA作为牺牲材料，3D打印得到模具；
(2)称取0.08g GelMA、0.01g HAMA、0.03g CSMA和2.5mg LAP，在37℃下溶于培养基中，将该混合物灌注于模具中，在405nm波长、强度10mW/cm 2的蓝光照射下实现光交联，即可获得8％GelMA/1％HAMA/3％CSMA固化水凝胶-模具复合体；
(3)将该固化水凝胶-模具复合体浸泡在二氯甲烷中，二氯甲烷溶解模具，即可获得8％GelMA/1％HAMA/3％CSMA固化水凝胶；
(4)将固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干8h，即可制备8％GelMA/1％HAMA/5％CSMA的仿生软骨支架。
实施例17
称取0.08g GelMA、0.01g HAMA、0.03g CSMA、0.02g海藻酸钠(Alg)和2.5mg LAP，在37℃下溶于1mL培养基中，将该混合物置于蓝光辅助挤出式3D打印机中，在405nm波长、强度10mW/cm 2的蓝光照射下3D打印，即可获得8％GelMA/1％HAMA/3％CSMA/2％Alg固化水凝胶。然后，将制备的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干20h，即可制备8％GelMA/1％HAMA/3％CSMA/2％Alg的仿生软骨支架。
实施例18
称取0.08g GelMA、0.01g HAMA、0.03g CSMA、0.1g PEGDA和0.01g LAP，在37℃下溶于1mL培养基中，将该混合物灌注于预制的硅胶成型模具中，在405nm波长、 强度10mW/cm 2的蓝光照射下实现光交联，即可获得8％GelMA/1％HAMA/3％CSMA/10％PEGDA固化水凝胶。然后，将制备的固化水凝胶放置于-20℃冰箱中预冷2h，再用冻干机冻干20h，即可制备8％GelMA/1％HAMA/3％CSMA/10％PEGDA的仿生软骨支架。
对比例1
称取0.05g GelMA、0.02g HAMA、0.32g CSMA和2.5mg LAP，在37℃下溶于1mL 0.9％NaCl溶液(pH＝7.4)中，发现CSMA含量过高会影响水凝胶机械性能。
对比例2
称取0.55g GelMA、0.02g HAMA、0.02g CSMA和2.5mg LAP，在37℃下溶于1mL 0.9％NaCl溶液(pH＝7.4)中，发现GelMA难以完全溶解，无法继续制备水凝胶。
对比例3
称取0.05g GelMA、0.35g HAMA、0.02g CSMA和2.5mg LAP，在37℃下溶于1mL 0.9％NaCl溶液(pH＝7.4)中，发现HAMA难以完全溶解，无法继续制备水凝胶。
效果实施例4：水凝胶的流变分析
称取0.05g GelMA、0.02g HAMA、0.02g CSMA和2.5mg LAP，在37℃下溶于1mL 0.9％NaCl溶液(pH＝7.4)中。在25℃时，使用一块平行板(P20TiL，20mm直径)在Haake Mars旋转流变仪上进行动态流变学实验。在蓝光(405nm，30mW/cm 2)照射下，以10％应变(CD模式)、1Hz频率和0.5mm间隙进行60s的扫描测试。模量随时间的变化如图29所示，凝胶点为储能模量(G’)超过损耗模量(G”)时的时间，可知凝胶点为3s。在10s左右储能模量达到最大，说明固化非常迅速，储能模量数值持续保持至测试结束，表明形成的凝胶结构稳定。
效果实施例5：水凝胶的力学性能
配制5％GelMA+1％HAMA+1％CSMA+0.25％LAP溶液，放入直径10mm、高度8mm的圆柱体模具中，在405nm波长的蓝光照射下实现光交联，即得5％GelMA+1％HAMA+1％CSMA固化水凝胶支架，作为测试样品。用GT-TCS-2000型单柱仪对固化水凝胶支架的力学性能进行测试，压缩速度设定为1mm/min，至压碎停止，得到应力-应变曲线，如图30所示。固化水凝胶支架破碎前的最大压强即为极限应力，弹性模量根据应力-应变曲线的15％～20％的斜率计算获得。由图30可知，该固化水凝胶支架的极限应力为170.8kPa，弹性模量为123.7kPa。
效果实施例6：仿生软骨支架的细胞相容性测试
称取0.05g GelMA、0.02g HAMA和0.02g CSMA，与含有P2代兔软骨细胞的培养 基混合，加入2.5mg LAP，在蓝光405nm照射下，5秒固化成胶，培养7天后，用Calcein/AM活死细胞染色试剂盒检测细胞存活情况，结果见图31，细胞在水凝胶中的存活率>90％。
效果实施例7：骨软骨缺损修复试验
1、骨软骨支架的制备：
(1)按照实施例13的方法制备5％GelMA/2％HAMA/2％CSMA的仿生软骨支架。
(2)称取10g聚乳酸(PLA)溶于20mL二氯甲烷后至完全溶解，加入1g羟基磷灰石(HAP)，搅拌至均匀溶液，然后置于通风橱待二氯甲烷挥发后，放入真空干燥箱烘干24h，即可制成为HAP/PLA高分子材料。将HAP/PLA高分子材料采用熔融沉积式3D打印机打印成型(打印头温度：210℃；平台温度：50℃；打印速度：60mm/s；填充率：50％)，得到HAP/PLA骨层。
(3)用医用胶水(金象)将仿生软骨支架和HAP/PLA骨层连接，即得骨软骨支架。
2、骨软骨缺损修复试验
取新西兰白兔15只，雌性，体重3～3.5kg，5月龄，骨骼成熟，在兔股骨滑车和内髁处钻孔，制造骨软骨缺损模型。在内髁缺损处植入上述骨软骨支架，作为实验组；软骨层周边填充GelMA基软骨水凝胶，滑车处不处理，作为空白对照。12周后安乐死，进行大体观察(见图32)和组织学检测(见图33)。如图32所示，植入了骨软骨支架的内髁处术后3个月，可以看到光滑的新生软骨，并与周围正常软骨连接良好，融为一体。如图33所示，空白对照的滑车处的软骨缺损严重，依然是一个空坑。图33的组织学分析还表明，内髁缺损处有软骨细胞陷窝生成，而滑车空白对照处无软骨细胞陷窝；番红O染色软骨基质呈红色，甲苯胺蓝染色呈紫红色，说明内髁缺损处分泌了新的软骨基质，且软骨下观察到松质骨的生成；而空白对照滑车处无软骨基质分泌，也没有松质骨的生成。
虽然以上描述了本发明的具体实施方式，但本领域技术人员应当理解，这些仅是举例说明，在不背离本发明原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (20)

  1. 一种仿生组织支架的制备方法，其包括以下步骤：
    S1、将牺牲材料进行3D打印，得到模具；其中，所述牺牲材料为可溶于溶剂的硬质高分子材料；
    S2、向所述模具中灌注水凝胶组合物，使其原位交联固化，得到固化水凝胶-模具复合体；
    S3、脱模：用溶剂溶解所述固化水凝胶-模具复合体中的模具，得到固化水凝胶；
    S4、将所得固化水凝胶冷冻干燥即得所述仿生组织支架。
  2. 如权利要求1所述的仿生组织支架的制备方法，其特征在于，
    所述牺牲材料为生物相容的；和/或，所述牺牲材料为透明的或半透明的；
    和/或，所述牺牲材料为聚乳酸、聚己内酯、聚对苯二甲酸乙二醇酯-1,4-环己烷二甲醇酯、聚乙烯醇或合成类光敏树脂；所述合成类光敏树脂较佳地为聚丙烯酸酯类光敏树脂；
    和/或，步骤S1中，先将颜料混于所述牺牲材料中，然后将带颜色的牺牲材料进行3D打印，得到带颜色的模具；
    和/或，步骤S1中，所述3D打印的方式为挤出方式或光固化方式；所述光固化方式较佳地为立体光固化成型技术、数字光投影技术或液晶显示技术。
  3. 如前述权利要求中至少一项所述的仿生组织支架的制备方法，其特征在于，步骤S2中，所述水凝胶组合物的可凝胶成分包括甲基丙烯酰化明胶、甲基丙烯酰化胶原、甲基丙烯酰化弹力蛋白、甲基丙烯酰化透明质酸、甲基丙烯酰化硫酸软骨素、甲基丙烯酰化海藻酸钠、甲基丙烯酰化肝素、明胶、胶原、弹力蛋白、透明质酸、硫酸软骨素、肝素和海藻酸钠中的一种或几种的组合；更佳地，所述可凝胶成分包括甲基丙烯酰化明胶、甲基丙烯酰化透明质酸和甲基丙烯酰化硫酸软骨素中的一种或多种；
    较佳地，所述仿生组织支架为软骨支架，所述可凝胶成分包括海藻酸钠和甲基丙烯酰化明胶；
    较佳地，所述仿生组织支架为软骨支架，所述可凝胶成分包括甲基丙烯酰化明胶和甲基丙烯酰化透明质酸；
    较佳地，所述仿生组织支架为软骨支架，所述可凝胶成分包括甲基丙烯酰化明胶、甲基丙烯酰化透明质酸和甲基丙烯酰化硫酸软骨素；
    较佳地，所述仿生组织支架为骨支架，所述可凝胶成分包括海藻酸钠、甲基丙烯酰化明胶和羟基磷灰石；
    较佳地，所述仿生组织支架为神经导管支架，所述可凝胶成分成分包括海藻酸钠和甲基丙烯酰化明胶；
    较佳地，所述仿生组织支架为皮肤支架，所述可凝胶成分成分包括甲基丙烯酰化胶原和甲基丙烯酰化明胶；
    较佳地，所述仿生组织支架为肌肉支架，所述可凝胶成分成分包括甲基丙烯酰化透明质酸和甲基丙烯酰化明胶；
    和/或，所述水凝胶组合物的凝胶介质为纯化水、生理盐水、细胞培养基、钙盐溶液和磷酸盐缓冲溶液中的一种或多种；
    和/或，所述水凝胶组合物还包括光引发剂；所述光引发剂较佳地为蓝光引发剂、紫外光引发剂或绿光引发剂；所述蓝光引发剂较佳地为苯基-2,4,6-三甲基苯甲酰基膦酸锂、核黄素、黄素单核苷酸、曙红Y或三联吡啶氯化钌/过硫酸钠；所述紫外光引发剂较佳地为2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮；
    和/或，所述水凝胶组合物还包括增稠剂；所述增稠剂较佳地为聚环氧乙烷、聚乙二醇、海藻酸钠、透明质酸、聚乙烯吡咯烷酮、阿拉伯树胶、结冷胶和黄原胶中的一种或多种；
    和/或，所述水凝胶组合物还包括合成类光敏材料；所述合成类光敏材料较佳地包括聚乙二醇丙烯酸酯、聚丙烯酸、聚甲基丙烯酸、聚丙烯酸酯、聚甲基丙烯酸酯、聚丙烯酰胺和聚甲基丙烯酰胺中的一种或多种；所述合成类光敏材料较佳地为聚乙二醇丙烯酸酯。
  4. 如前述权利要求中至少一项所述的仿生组织支架的制备方法，其特征在于，
    步骤S2中，所述交联固化的方式包括物理交联固化、化学交联固化和光交联固化中的一种或多种；较佳地包括光交联固化；其中，所述光交联固化较佳地在波长365～405nm、强度5～50mW/cm 2的光照射下进行；更佳地，所述光交联在波长405nm、强度10mW/cm 2的光照射下进行；
    和/或，步骤S3中，所述溶剂为二氯甲烷、三氯甲烷、四氢呋喃、1,4-二氧六环、纯化水、生理盐水、钙盐溶液、磷酸盐缓冲溶液或培养基；
    和/或，步骤S4中，所述冷冻干燥的时间为8～24h；所述冷冻干燥之前较佳地还进行预冷步骤；所述预冷的温度较佳地为-20℃，所述预冷的时间较佳地为1～3h。
  5. 一种仿生组织支架，其根据权利要求1～4中任一项所述的仿生组织支架的制备方法制得。
  6. 一种骨软骨支架，其包括软骨层、黏连层和骨层，所述黏连层的两侧分别与所述软骨层和所述骨层连接；所述软骨层、所述黏连层和所述骨层中的一种或多种为多孔结 构。
  7. 如权利要求6所述的骨软骨支架，其特征在于，所述软骨层、所述黏连层和所述骨层均为多孔结构；较佳地，所述软骨层的孔、所述黏连层的孔与所述骨层的孔连通；所述软骨层的孔、所述黏连层的孔与所述骨层的孔可完全对齐或不完全对齐，较佳地为完全对齐；
    和/或，所述黏连层不覆盖或部分覆盖所述软骨层和/或所述骨层的孔；
    和/或，所述软骨层和/或所述骨层的孔的孔径为50～350μm，较佳地为200～280μm，例如250μm；较佳地，所述骨层的孔的孔径与所述软骨层的孔的孔径相等；
    和/或，所述软骨层和/或所述骨层的孔的分布方式较佳地为垂直交叉排列；
    和/或，所述软骨层和/或所述骨层的孔隙率为20％～70％，较佳地为40％～60％，例如50％；
    和/或，所述骨软骨支架的孔隙率为20％～70％，较佳地为40％～60％，例如50％；
    和/或，所述骨软骨支架为圆柱体；所述圆柱体的直径较佳地为2～20mm，更佳地为3～10mm；所述圆柱体的高度较佳地为2～10mm，更佳地为3～6mm；或者，所述骨软骨支架为长方体；所述长方体的底面可为正方形，所述正方形的边长可为2～30mm，较佳地为2～20mm，更佳地为3～10mm；所述长方体的高度较佳地为2～10mm，更佳地为3～6mm；
    和/或，所述骨层和所述软骨层的高度比为1：(0.1～1)，较佳地为1：(0.2～0.5)；
    和/或，所述黏连层的高度为5μm～2mm，较佳地为0.1～2mm，更佳地为0.5～1mm；
    和/或，所述软骨层的材料为水凝胶材料；
    和/或，所述软骨层负载软骨促进成分；其中，所述软骨促进成分较佳地包括生物活性因子和/或细胞，所述生物活性因子较佳地包括转化生长因子TGFα或TGFβ，所述细胞较佳地包括自体或异体软骨细胞、间充质干细胞、胚胎干细胞或诱导性多能干细胞；
    和/或，所述骨层的材料为聚乳酸、聚乳酸-羟基乙酸共聚物或聚己内酯；
    和/或，所述骨层负载骨促进成分；其中，所述骨促进成分较佳地包括生物活性的无机材料、生物活性因子和细胞中的一种或多种；
    其中，所述生物活性的无机材料较佳地包括羟基磷灰石、磷酸钙、碳酸钙和生物活性玻璃中的一种或多种；所述生物活性的无机材料在所述骨层中的质量百分比可为0.1wt％～70wt％，较佳地为1wt％～50wt％，更佳地为2.5wt％～30wt％；
    所述生物活性因子较佳地包括转化生长因子TGFα、TGFβ，骨形态发生蛋白BMP-2、BMP-3、BMP-4、BMP-5、BMP-6、BMP-7、BMP-8和BMP-9中的一种或多种；
    所述细胞较佳地包括自体或异体骨细胞、间充质干细胞、胚胎干细胞或诱导性多能干细胞；
    和/或，所述黏连层由医用胶水形成；或者，所述黏连层的材料为水凝胶材料。
  8. 一种如权利要求6或7所述的骨软骨支架的制备方法，其包括以下步骤：将骨层和软骨层连接，连接处形成黏连层；所述软骨层、所述黏连层和所述骨层中的一种或多种为多孔结构。
  9. 如权利要求8所述的骨软骨支架的制备方法，其特征在于，所述软骨层的制备方法为以水凝胶组合物为原料经交联固化制备；其中，所述水凝胶组合物至少包括可凝胶成分和凝胶介质；
    所述可凝胶成分较佳地包括天然可凝胶成分和/或合成可凝胶成分；其中，所述天然可凝胶成分较佳地包括天然蛋白、天然蛋白修饰物、天然蛋白降解物、天然蛋白降解物的修饰物、天然多糖、天然多糖修饰物、天然多糖降解物和天然多糖降解物的修饰物中的一种或多种；所述天然蛋白较佳地包括各种亲水动植物蛋白、水溶性动植物蛋白、I型胶原蛋白、II型胶原蛋白、血清蛋白、丝素蛋白和弹性蛋白中的一种或多种；所述天然蛋白降解物较佳地包括明胶或多肽；所述天然蛋白降解物的修饰物较佳地为甲基丙烯酰化天然蛋白降解物，更佳地为甲基丙烯酰化明胶；所述天然多糖较佳地包括透明质酸、羧甲基纤维素、甲基纤维素、羟乙基纤维素、羟丙基纤维素、海藻酸、葡聚糖、琼脂糖、肝素、硫酸软骨素、乙二醇壳聚糖、丙二醇壳聚糖、壳聚糖乳酸盐、羧甲基壳聚糖和壳聚糖季铵盐中的一种或多种，更佳地为透明质酸和/或硫酸软骨素；所述天然多糖修饰物较佳地为甲基丙烯酰化天然多糖，例如甲基丙烯酰化透明质酸或甲基丙烯酰化硫酸软骨素；
    所述合成可凝胶成分较佳地包括两臂或多臂聚乙二醇双丙烯酸酯、聚乙烯亚胺、合成多肽、聚丙烯酸、聚甲基丙烯酸、聚丙烯酸酯、聚甲基丙烯酸酯、聚丙烯酰胺、聚甲基丙烯酰胺、聚乙烯醇和聚乙烯吡咯烷酮中的一种或多种；
    较佳地，所述可凝胶成分包括甲基丙烯酰化明胶和甲基丙烯酰化透明质酸；更佳地，所述水凝胶的可凝胶成分包括甲基丙烯酰化明胶、甲基丙烯酰化透明质酸和甲基丙烯酰化硫酸软骨素；较佳地，所述软骨层的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶1～50份，甲基丙烯酰化透明质酸0～30份，甲基丙烯酰化硫酸软骨素0～30份，光引发剂0.01～1份和凝胶介质；
    其中，所述甲基丙烯酰化明胶的用量较佳地为1～30份，更佳地为1～20份，更佳地为2～15份，更佳地为5～15份，例如8份、10份或12份；
    其中，所述甲基丙烯酰化透明质酸的用量较佳地为0.1～20份，更佳地为0.5～10份， 更佳地为1～3份，例如1.5份或2份；
    其中，所述甲基丙烯酰化硫酸软骨素的用量较佳地为0.1～20份，更佳地为0.5～20份，更佳地为0.5～5份，更佳地为1～3份，例如1份、2份、2.5份或3份；
    其中，所述甲基丙烯酰化明胶和所述甲基丙烯酰化透明质酸的质量比可为(1-30)：(0.5～10)，较佳地为(2～15)：(1～3)，例如5:2；
    其中，所述甲基丙烯酰化明胶、所述甲基丙烯酰化透明质酸和所述甲基丙烯酰化硫酸软骨素的质量比可为(1～30)：(0.5～10)：(0.5～20)，较佳地为(2～15)：1：(1～3)，例如10:1:3，5:2:2，15:1:1或8:1:3；
    可选地，所述软骨层的水凝胶组合物还包括增稠剂，所述增稠剂的用量较佳地为0.1～25份；
    可选地，所述软骨层的水凝胶组合物还包括合成类光敏材料，所述合成类光敏材料的用量较佳地为5～30份；
    较佳地，所述凝胶介质为纯化水、生理盐水、细胞培养基、钙盐溶液和磷酸盐缓冲溶液中的一种或多种；所述凝胶介质的用量较佳地使得所述水凝胶组合物中：甲基丙烯酰化明胶5％～30％，甲基丙烯酰化透明质酸0.5～2％，甲基丙烯酰化硫酸软骨素0.1％～5％，光引发剂0.01～1％；其中百分比为每100mL凝胶介质含有的组分质量(g)；
    较佳地，所述光引发剂为蓝光引发剂、紫外光引发剂或绿光引发剂；所述蓝光引发剂较佳地为苯基-2,4,6-三甲基苯甲酰基膦酸锂、核黄素、黄素单核苷酸、曙红Y或三联吡啶氯化钌/过硫酸钠；所述紫外光引发剂较佳地为2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮；
    其中，所述交联固化的方式较佳地包括物理交联、化学交联、酶促交联和光交联中的一种或多种；其中，所述光交联较佳地在波长365～405nm、强度5～50mW/cm 2的光照射下进行；所述光交联更佳地在波长405nm、强度10mW/cm 2的光照射下进行；
    较佳地，所述软骨层的制备方法为灌注法，其包括以下步骤：将所述软骨层的水凝胶组合物灌注于软骨模具中进行光交联，得到固化水凝胶；将所述固化水凝胶冷冻干燥即得；
    较佳地，所述软骨层的制备方法为直接3D打印法，其包括以下步骤：将所述软骨层的水凝胶组合物进行3D打印同时进行光交联，得固化水凝胶；将所述固化水凝胶冷冻干燥即得；更佳地，所述软骨层的制备方法包括以下步骤：将所述软骨层的水凝胶组合物采用挤出式3D打印机打印，蓝光固化成型，其中，保温温度为30～37℃，打印环境温度为22～25℃，打印压力为20～40PSI，打印速度为4～8mm/s，填充率为40％～60％，光照强 度为5～20mW/cm 2；
    较佳地，所述软骨层的制备方法为3D打印脱模法，其包括以下步骤：
    S1、将牺牲材料进行3D打印，得到软骨层模具；其中，所述牺牲材料为可溶于溶剂的硬质高分子材料；
    S2、将软骨层的水凝胶组合物灌注于所述软骨层模具中进行交联，得到固化水凝胶-软骨层模具复合体；
    S3、脱模：用溶剂溶解所述软骨层模具，得到固化水凝胶；
    S4、将所得固化水凝胶冷冻干燥即得所述软骨层；
    和/或，所述软骨层的制备方法还包括负载软骨促进成分的步骤。
  10. 如权利要求8或9所述的骨软骨支架的制备方法，其特征在于，所述骨层的制备方法为对所述骨层的材料进行3D打印；所述3D打印较佳地采用熔融沉积式3D打印机进行；所述骨层的材料较佳地为聚乳酸、聚乳酸-羟基乙酸共聚物或聚己内酯；
    较佳地，所述骨层通过采用熔融沉积式3D打印机对负载羟基磷灰石的PLA进行3D打印来提供，其中，打印头温度为210℃，平台温度为50～60℃，打印速度为50～60mm/s，填充率为40％～60％；
    较佳地，所述骨层通过采用熔融沉积式3D打印机对负载有磷酸三钙的PCL进行3D打印来提供，其中，打印温度为140～150℃，打印速度为50～60mm/s，填充率为40％～60％；
    较佳地，所述骨层通过采用挤出式光固化3D打印机对负载有磷酸三钙的GelMA进行3D打印来提供，其中，保温温度为30-35℃，打印环境温度为22～25℃，打印压力为20～40PSI，打印速度为4-8mm/s，填充率为40-60％，光照强度为5～50mW/cm 2；
    和/或，所述骨层的制备方法为以水凝胶组合物为原料经交联固化制备；
    较佳地，所述骨层的水凝胶组合物包括以下组分：甲基丙烯酰化明胶5％～30％，生物活性玻璃2.5％～50％和光引发剂0.01％～1％；其中百分比为每100mL凝胶介质含有的组分质量(g)；
    和/或，所述骨层的制备方法还包括负载骨促进成分的步骤；
    所述负载骨促进成分的较佳方法包括：将所述骨层的材料磨成粉末后掺入所述骨促进成分；或者，将所述骨层的材料溶于溶剂后掺入所述骨促进成分，然后使所述溶剂挥发即可。
  11. 如权利要求8或9所述的骨软骨支架的制备方法，其特征在于，所述骨软骨支架采用3D打印脱模法，其包括以下步骤：
    S1、将牺牲材料进行3D打印，得到骨软骨支架模具；其中，所述牺牲材料为可溶于 溶剂的硬质高分子材料；
    S2、依次将骨层、黏连层和软骨层的水凝胶组合物灌注于所述骨软骨支架模具中进行交联，得到固化水凝胶-骨软骨支架模具复合体；
    S3、脱模：用溶剂溶解所述骨软骨支架模具，得到固化水凝胶；
    S4、将所得固化水凝胶冷冻干燥即得所述骨软骨支架；
    较佳地，所述骨层的水凝胶组合物包括以下组分：甲基丙烯酰化明胶5％～30％，生物活性玻璃2.5％～50％和光引发剂0.01％～1％；所述黏连层的水凝胶组合物包括以下组分：甲基丙烯酰化明胶5％～30％，生物活性玻璃10％～20％和光引发剂0.01％～1％；所述软骨层的水凝胶组合物包括以下组分：甲基丙烯酰化明胶5％～30％，甲基丙烯酰化透明质酸0.5％～2％，甲基丙烯酰化硫酸软骨素0.5％～5％，和光引发剂0.01％～1％；其中，百分比为每100mL凝胶介质含有的组分质量(g)。
  12. 一种如权利要求6或7所述的骨软骨支架在修复骨软骨缺损中的应用。
  13. 一种用于仿生软骨支架的水凝胶组合物，其包括按质量份数计的以下组分：甲基丙烯酰化明胶1～50份，甲基丙烯酰化透明质酸0.1～30份，甲基丙烯酰化硫酸软骨素0.1～30份，光引发剂0.01～1份和凝胶介质。
  14. 根据权利要求13所述的用于仿生软骨支架的水凝胶组合物，其特征在于，所述甲基丙烯酰化明胶的用量为1～20份，较佳地为5～15份，例如8份、10份或12份；
    和/或，所述甲基丙烯酰化明胶的甲基丙烯酰化程度为30％～100％，较佳地为40％～80％；
    和/或，所述甲基丙烯酰化透明质酸的用量为0.1～10份，较佳地为0.5～10份，更佳地为0.5～2份，例如0.5份、1份、1.5份；
    和/或，所述甲基丙烯酰化透明质酸的分子量为1～8000kDa，较佳地为100～1000kDa，更佳地为500～950kDa；
    和/或，所述甲基丙烯酰化透明质酸的甲基丙烯酰化程度为20％～60％，较佳地为30％～50％；
    和/或，所述甲基丙烯酰化硫酸软骨素的用量为0.1～10份，较佳地为0.5～10份，更佳地为0.5～3份，例如1份、2份或2.5份；
    和/或，所述甲基丙烯酰化硫酸软骨素的分子量为5～50kDa，较佳地为10～40kDa；
    和/或，所述甲基丙烯酰化硫酸软骨素的甲基丙烯酰化程度为20％～60％，较佳地为30％～50％；
    和/或，所述甲基丙烯酰化明胶、所述甲基丙烯酰化透明质酸和所述甲基丙烯酰化硫 酸软骨素的质量比为(2-15)：(0.5-5)：(1-5)，更佳地为(2-15)：1：(1-3)，例如10:1:3，5:2:2，15:1:1或8:1:3；
    和/或，所述光引发剂的用量为0.1～0.5份，例如0.25份。
  15. 根据权利要求13或14所述的用于仿生软骨支架的水凝胶组合物，其特征在于，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶5～15份，甲基丙烯酰化透明质酸0.5～10份，甲基丙烯酰化硫酸软骨素0.5～10份，和光引发剂0.1～0.5份；
    较佳地，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶5份，甲基丙烯酰化透明质酸2份，甲基丙烯酰化硫酸软骨素2份，和光引发剂0.25份；
    较佳地，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶10份，甲基丙烯酰化透明质酸1份，甲基丙烯酰化硫酸软骨素3份，和光引发剂0.25份；
    较佳地，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶15份，甲基丙烯酰化透明质酸1份，甲基丙烯酰化硫酸软骨素1份，和光引发剂0.25份；
    较佳地，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶8份，甲基丙烯酰化透明质酸1份，甲基丙烯酰化硫酸软骨素3份，和光引发剂0.25份；
    较佳地，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶8份，甲基丙烯酰化透明质酸1份，甲基丙烯酰化硫酸软骨素3份，海藻酸钠2份，和光引发剂0.25份；
    较佳地，所述用于仿生软骨支架的水凝胶组合物包括按质量份数计的以下组分：甲基丙烯酰化明胶8份，甲基丙烯酰化透明质酸1份，甲基丙烯酰化硫酸软骨素3份，聚乙二醇丙烯酸酯10份，和光引发剂1份。
  16. 根据权利要求13～15中至少一项所述的用于仿生软骨支架的水凝胶组合物，其特征在于，所述用于仿生软骨支架的水凝胶组合物还包括增稠剂，所述增稠剂的用量较佳地为0.1～25份；其中，当所述增稠剂包括海藻酸钠时，所述海藻酸钠的用量较佳地为1～2份；当所述增稠剂包括透明质酸时，所述透明质酸的用量较佳地为0.5～2份；当所述增稠剂包括聚乙烯吡咯烷酮时，所述聚乙烯吡咯烷酮的用量较佳地为2～10份；当所述增稠剂包括阿拉伯树胶时，所述阿拉伯树胶的用量较佳地为0.1～25份；当所述增稠剂包括 结冷胶时，所述结冷胶的用量较佳地为0.1～2份；当所述增稠剂包括黄原胶时，所述黄原胶的用量较佳地为0.1～1份；
    和/或，所述用于仿生软骨支架的水凝胶组合物还包括合成类光敏材料；所述合成类光敏材料的用量较佳地为5～30份；
    和/或，所述凝胶介质的用量使得所述用于仿生软骨支架的水凝胶组合物中：甲基丙烯酰化明胶5％-20％，甲基丙烯酰化透明质酸0.1％～3％，甲基丙烯酰化硫酸软骨素0.1％～5％和光引发剂0.01％～1％；其中百分比为每100mL凝胶介质含有的组分质量(g)。
  17. 一种仿生软骨支架的制备方法，其采用权利要求13～16中任一项所述的用于仿生软骨支架的水凝胶组合物为原料，经光交联固化得到。
  18. 根据权利要求17所述的仿生软骨支架的制备方法，其特征在于，所述仿生软骨支架的制备方法为3D打印脱模法，其包括以下步骤：
    S1、将牺牲材料进行3D打印，得到仿生软骨支架模具；其中，所述牺牲材料为可溶于溶剂的硬质高分子材料；
    S2、将软骨层的水凝胶组合物灌注于所述仿生软骨支架模具中进行交联，得到固化水凝胶-仿生软骨支架模具复合体；
    S3、脱模：用溶剂溶解所述仿生软骨支架模具，得到固化水凝胶；
    S4、将所得固化水凝胶冷冻干燥即得所述仿生软骨支架；
    或者，所述仿生软骨支架的制备方法为灌注法或直接3D打印法。
  19. 一种仿生软骨支架，其由权利要求17或18所述仿生软骨支架的制备方法制得。
  20. 一种所述用于仿生软骨支架的水凝胶组合物或所述仿生软骨支架在骨软骨组织工程中的应用。
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