WO2021012677A1 - 一种仿生的预脉管化材料及其制备方法和应用 - Google Patents

一种仿生的预脉管化材料及其制备方法和应用 Download PDF

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WO2021012677A1
WO2021012677A1 PCT/CN2020/077831 CN2020077831W WO2021012677A1 WO 2021012677 A1 WO2021012677 A1 WO 2021012677A1 CN 2020077831 W CN2020077831 W CN 2020077831W WO 2021012677 A1 WO2021012677 A1 WO 2021012677A1
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vascularized
vein
materials
pdms
cells
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PCT/CN2020/077831
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French (fr)
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张利军
邢孟秋
吴军
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中山大学附属第一医院
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    • 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
    • A61L27/222—Gelatin
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3808—Endothelial cells
    • 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
    • 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
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus

Definitions

  • the present invention relates to the technical field of biomedicine, and more specifically, to a biomimetic pre-vascularized material and its preparation method and application.
  • vascularization is a necessary condition for wound repair. Early vascularization can improve graft survival rate, wound repair efficiency and quality. It has been proved that the pre-vascularized material can establish blood circulation 3 days earlier than the non-pre-vascularized material. It can be seen that the preparation and application of the pre-vascularized material has a strong necessity and good application effect.
  • the methods and means for repairing lymphatic damage and improving lymphatic function have not been significantly improved. Therefore, it is necessary and important to explore a way to repair the function and structure of lymphatic vessels.
  • the pre-vascularization materials and preparation methods used mainly include: using scaffold materials, such as acellular matrix, composite seed cells (stem cells, vascular endothelial cells) and other in vitro culture to obtain pre-vascularization materials; or using cell patch technology
  • scaffold materials such as acellular matrix, composite seed cells (stem cells, vascular endothelial cells) and other in vitro culture to obtain pre-vascularization materials
  • cell patch technology For example, Professor Okano and others used temperature-sensitive materials to culture cell membranes in vitro, and composite vascular endothelial cells to obtain pre-vascularized materials with 3D structure, which have now been used in the repair of cornea, esophagus and other tissues and organs.
  • the preparation method of pre-lymphatic materials is mainly the technology of mixing cells extracted from skin tissues and culturing in vitro to obtain materials for lymphatics and blood vessels.
  • the general feature of the above technology is the use of biological, chemical, material science or engineering techniques to prepare scaffold materials and composite vascular endothelial cells to construct vascularized materials.
  • biotechnological methods need to select appropriate allogeneic or heterogeneous tissues, and use chemical or physical methods to decellularize to obtain matrix materials.
  • the entire process is relatively complicated and time-consuming, and it involves ethical and rejection issues in clinical applications.
  • In vitro culturing of cell membranes requires technology and a platform for preparing temperature-sensitive materials.
  • In vitro culturing of 3D vascularized materials with sufficient tissue volume requires multi-layer membrane stacking, which has relatively complicated technical means and many operating procedures;
  • Materials involve issues such as material modification, microstructure, structure description, degradation rate and toxicity of degradation products.
  • the mixed cell culture method of skin tissue has a long cell extraction and culture cycle, and the obtained tissue thickness is insufficient, which is not suitable for large-area skin or tissue repair.
  • the technical problem solved by the present invention is to overcome the existing problems and provide a bionic pre-vascularized material
  • Another object of the present invention is to provide a method for preparing a biomimetic pre-vascularized material
  • Another object of the present invention is to provide an application of a biomimetic pre-vascularized material in the preparation of wound surface and tissue repair materials, scaffold materials, organ reconstruction materials, tissue filling materials or wrapping materials.
  • a method for preparing a bionic pre-vascularized material includes the following steps:
  • Step 2 After disinfecting and washing the biological material obtained in step 1, place it in a cell culture medium and let it stand overnight, and then inoculate cells for in vitro cell culture;
  • the biological material includes but is not limited to: (1) natural polysaccharides, such as cellulose, chitin, chitosan, lentinan; (2) amphoteric polysaccharide derivatives, such as polylactic acid, polycaprolactone and the like Derivatives; (3) Natural polymer hydrogels, such as chitosan hydrogel; (4) GelMA, gelatin, hyaluronic acid and their improved materials.
  • natural polysaccharides such as cellulose, chitin, chitosan, lentinan
  • amphoteric polysaccharide derivatives such as polylactic acid, polycaprolactone and the like Derivatives
  • Natural polymer hydrogels such as chitosan hydrogel
  • GelMA gelatin, hyaluronic acid and their improved materials.
  • step 1. includes the following steps:
  • Step 11 Place the vein structure of natural plants in a petri dish or other container, with the back side facing up, and moisten it to make it fit with the bottom surface of the container;
  • Step 12 Polydimethylsiloxane film formation: prepare PDMS and cover the surface of the vein structure in step 1 for curing, then remove the veins to obtain a PDMS film with a vein "mirror structure";
  • Step 13 Polydimethylsiloxane inverted mold: Prepare PDMS and lay it flat on the surface of the PDMS film with veins "mirror structure" in step 2, after curing, remove the PDMS with veins "mirror structure” Film to obtain PDMS template with the same structure as leaf veins;
  • Step 14 Take GelMA solution or gelatin on the surface of the PDMS template obtained in step 3 for cross-linking and curing, and then remove the PDMS template to obtain GelMA or gelatin with a vein structure.
  • step 1 includes the following steps:
  • biological materials are used as "printing ink” and the vein structure is used as a template to print biological materials with vein structure.
  • step 1 includes the following steps:
  • the biological material with leaf vein structure is printed.
  • the pre-vascularization includes pre-vascularization and pre-lymphaticization.
  • the invention discloses a pre-vascularized tissue engineering material prepared by using bionic technology, which is composed of a biocompatible absorbable scaffold and one or more cells, and uses or does not use bioreactors and microfluidic equipment Processing and cultivation.
  • Such pre-vascularized materials can be used to repair or fill damaged or diseased tissues.
  • the speed and efficiency of vascularization or vascularization affect the survival rate of the graft and its tissue filling and repair effect.
  • Pre-vascularized materials can significantly shorten this process.
  • the biomimetic technology used in the present invention utilizes the similarity of the shape of the veins to the vascularized structure of the tissue microcirculation, and uses the method of "topology, 3D printing technology, laser printing technology, etc.” to use biocompatible Biological materials (including but not limited to GelMA or gelatin, etc.) and cell culture technology to obtain pre-vascularized materials.
  • biocompatible Biological materials including but not limited to GelMA or gelatin, etc.
  • cell culture technology to obtain pre-vascularized materials.
  • GelMA or gelatin with the surface of the vein structure functions as a scaffold material, which can be improved as needed to make it have mechanical properties suitable for predictable applications, and can maintain sufficient integrity until the tissue ingrowth and healing; it has the characteristics of easy processing and shaping into a variety of products and acceptable storage life; but it will not cause obvious inflammatory or toxic reactions in the body.
  • vein structure of the natural plant in step 11 is prepared by any of the following methods:
  • Method 1 Put the leaves in 8 ⁇ 12% sodium hydroxide solution and boil for 5 ⁇ 10 minutes, wash away the residual sodium hydroxide solution with running water, scrape the leaf flesh with tweezers or brush, and the veins can be obtained after washing;
  • Method 2 Put the leaves in water and boil for 5 ⁇ 10 minutes, then wash away the residual solution with running water, scrape gently with tweezers or brush to remove the leaf flesh, and the veins can be obtained after washing;
  • Method 3 Put the leaves in a mixed solution of 3 ⁇ 5% sodium hydroxide and 2 ⁇ 5% anhydrous sodium carbonate, boil for 5 ⁇ 10 minutes, wash away the corrosive solution in clean water, and then brush off the leaf flesh with a brush. Leaf veins can be obtained after washing.
  • the method for preparing PDMS is: mixing silicone and curing agent in a mixing ratio of 8-12:1, after removing bubbles, curing reaction at a temperature of 25-150° C. for 10 minutes to 24 hours.
  • the curing conditions of PDMS are: curing reaction at a temperature of 65° C. for 4 hours.
  • the thickness of the PDMS film in step 12 and step 13 is 1 mm to 10 mm; further preferably, the thickness of the PDMS film in step 12 and step 13 is 2 mm to 5 mm;
  • the method for removing bubbles includes vacuuming and centrifugation.
  • the centrifugation method after mixing the silicone and the curing agent, centrifuge at 1500-3000 rpm/min for 5-30 minutes.
  • the crosslinking and curing in step 14 includes any of the following methods:
  • DPBS phosphate buffered saline
  • TEMED tetramethylethylenediamine
  • step 3 The surface of the obtained PDMS template which has been pre-cooled on ice or at 2-8°C is cross-linked and solidified.
  • the curing and molding conditions are standing at -20°C for 8-24 hours, and then reheating at 4°C for 4-24 hours.
  • the cross-linking curing molding conditions are irradiating under a UV lamp of 365nm and 10-20W for 10-60 seconds.
  • the disinfection method in step 2 includes any of the following methods:
  • GelMA or gelatin with leaf vein structure is placed in a sterile environment, soaked in 70%-75% alcohol at room temperature for 30 minutes, and then washed with sterile phosphate buffered saline (DPBS) or PBS for 1 to 5 times. Soak for 2 ⁇ 10 minutes once;
  • DPBS sterile phosphate buffered saline
  • UV disinfection or gas disinfection is performed on GelMA or gelatin with vein structure.
  • ultraviolet light sterilization is performed for 30 minutes; the gas sterilization is sterilization using ethylene oxide gas.
  • the cells and cell culture medium in step 2 include one or more of the following:
  • Vascular endothelial cells including umbilical vein endothelial cells, microvascular endothelial cells, and endothelial progenitor cells of autologous, allogeneic or xenogeneic origin;
  • Vascular endothelial cell complete medium endothelial cell basal medium, 5-10% FBS, 1% double antibody, vascular growth factor; the vascular growth factor includes factors or proteins that promote the division, proliferation and/or differentiation of endothelial cells;
  • Stem cells which include embryonic stem cells and adult stem cells of autologous, allogeneic or xenogeneic origin, adipose stem cells, bone marrow mesenchymal stem cells, epidermal stem cells, and neural stem cells;
  • Stem cell culture medium 10% FBS, ⁇ -MEM, mesenchymal stem cell medium (MSCM), high glucose medium (DMEM) and/or targeted differentiation medium, 1% double antibody;
  • Fibroblasts fibroblast culture medium of autologous, allogeneic or xenogeneic origin: DMEM, 10% FBS, 1% double antibody;
  • iPS cells Induced pluripotent stem cells (iPS cells) of autologous, allogeneic or xenogeneic origin, stem cell culture medium: Essential 6TM Medium;
  • Lymphatic endothelial cells of autologous, allogeneic or xenogeneic origin; Lymphatic endothelial cell complete medium: endothelial cell basal medium, 5-10% FBS, 1% double antibody, vascular growth factor; the vascular growth factor includes promoting Factors or proteins that divide, proliferate and differentiate endothelial cells.
  • the umbilical vein endothelial cells are allogeneic, and the microvascular endothelial cells and endothelial progenitor cells may be of autologous, allogeneic or xenogeneic origin.
  • the vascular growth factor includes: one or more of vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF), platelet growth factor (PDGF), and transforming growth factor ⁇ (TGF ⁇ ), A factor or protein that promotes the division, proliferation and/or differentiation of endothelial cells.
  • VEGF vascular endothelial cell growth factor
  • FGF fibroblast growth factor
  • PDGF platelet growth factor
  • TGF ⁇ transforming growth factor ⁇
  • a bionic pre-vascularized material prepared by the above preparation method.
  • biomimetic pre-vascularization material in preparation of wound surface and tissue repair materials, scaffold materials, organ reconstruction materials, tissue replacement materials, tissue filling materials or wrapping materials, and in-situ induction regeneration materials.
  • the pre-vascularized material constructed in the present invention can be used to repair solid tissues or organs, such as skin, breast, liver, and spleen; it can also be prepared to repair the esophagus, trachea, stomach, and intestine by improving the physical properties of the scaffold material.
  • Heart and other hollow organs in the inner membrane or outer layer repair can also be used for tissue filling or wrapping in the field of plastic surgery.
  • the biomimetic pre-vascularized material can be superimposed after cell seeding, or superimposed with the pre-vascularized material on the surface of the veinless structure to form a three-dimensional vascularized network structure with a hollow structure.
  • the wound surface and tissue repair materials, scaffold materials, organ reconstruction materials, tissue replacement materials, tissue filling materials or wrapping materials, and in-situ induction regeneration materials include: subcutaneous filling materials, dermal tissue atrophy or defect filling materials , Muscle tissue defect filling or replacement material, breast filling or replacement material, nasal filling or replacement material, hip filling or replacement material, or prosthesis wrapping material, artificial vascularized skin flap, vascularized artificial tissue, in-situ suture replacement material, One or more of the missing or inactivated tissue/organ materials.
  • the biomimetic pre-vascularized material prepared by the present invention has a reticular branch structure with leaf veins similar to the microcirculation network structure of the human body, and can establish blood circulation at least 3 days earlier in wound repair, thereby improving the survival rate of the graft.
  • the vein structure of natural plants used in the present invention is widely available and easy to obtain. Leaves from different tree species can be selected as structural templates according to the needs of vessel diameter in different organs and tissues.
  • the biomimetic technology used in the present invention innovatively utilizes the characteristic that the veins of leaves are similar to the microcirculation vascularized structure of the tissue, and uses the "extension model” method to prepare a PDMS template with the same structure as the veins.
  • the biological material including but not limited to GelMA or gelatin, etc.
  • the biological material functions as a scaffold material. It can be improved as needed, and the cross-linking scheme can be adjusted to make it suitable for predictable applications. Performance, can maintain sufficient integrity until the tissue ingrowth and heal; it has the characteristics of easy processing and shaping into a variety of products and acceptable storage life; but it will not cause obvious inflammation or toxicity in the body.
  • Figure 1 shows the vein structure of natural plants
  • Figure 2 is a polydimethylsiloxane film with a "mirror structure" of leaf veins
  • Figure 3 is a polydimethylsiloxane template with a vein structure
  • Figure 4 shows GelMA on the surface of the vein structure
  • Picture A in Fig. 5 is GelMA, and picture B is GelMA on the surface of the vein structure;
  • Picture A in Fig. 6 shows the immunofluorescence staining image after inoculation of vascular endothelial cells on the GelMA surface with the vein structure surface;
  • Panel B shows the immunofluorescence staining image after the surface of GelMA without vein structure surface is inoculated with vascular endothelial cells, the red is actin, Blue is DAPI.
  • Figure 7 is a photograph taken with a confocal laser microscope (Confocal, Leica, confocal laser microscope).
  • Figures 8 to 11 are respectively the immunofluorescence staining images of Examples 2 to 5, with actin in red, DAPI in blue, and superimposed images of red and blue.
  • Fig. 12 is a picture taken after 3 weeks of implanting a pre-vascularized material with a hollow structure and a three-dimensional vascularized structure in a nude mouse.
  • Figure 13 is a picture of a pre-vascularized material with a three-dimensional vascularized structure having a hollow structure implanted in nude mice for 12 weeks.
  • a method for preparing a biomimetic prevascularized material includes the following steps:
  • Step 1 Vein structure production: Choose leaves with reticulated veins, which are moderately mature, with pale yellow flesh and yellowish brown veins. Place the leaves in a 10% sodium hydroxide solution and boil for 10 minutes, then wash away the residual sodium hydroxide solution with running water, scrape the leaves with tweezers or brush to remove the flesh, leaving the whole vein structure, as shown in Figure 1;
  • the vein structure Place the vein structure in a petri dish or other container, moisten it to make it fit completely with the bottom surface of the container; the method of laying the vein: the back of the vein structure is facing up (that is, the back of the leaf is facing up), and the other side of the vein structure is facing the culture Dish
  • Step 2 PDMS film making: use commercially available Dow Corning SYLGARDTM 184 silicone elastomer and make PDMS according to its instructions. Mix the silicone and curing agent at 10:1, mix well, and centrifuge at 1500rpm/min for 30 minutes ; Pour the centrifuged PDMS into a petri dish to cover the surface of the vein structure in step 1, with a thickness of about 2mm, and place it on the tabletop, vacuum to reduce bubbles; react at 65°C for 4 hours after the PDMS solidifies. After returning to room temperature, remove the veins to obtain a PDMS film with veins "mirror structure", as shown in Figure 2;
  • Step 3 PDMS inverted mold: Prepare PDMS according to the method of step 2 again, and spread it on the surface of the PDMS film with leaf vein "mirror structure" in step 2, the covering thickness of PDMS is about 2mm, and the rest is the same as in step 2, and the leaf veins are obtained.
  • PDMS template with the same structure, as shown in Figure 3;
  • Step 4 Place the GelMA solution on the surface of the PDMS template obtained in step 3 for cross-linking and curing, and then remove the PDMS template to obtain GelMA with a vein structure, as shown in Figures 4 and 5;
  • DPBS solution was used to prepare 5% GelMA, mixed with APS and TEMED, and quickly spread on the surface of the PDMS template obtained in step 3 for cross-linking and curing, and then let it stand at -20°C for 12 hours, and reheat at 4°C. 24 hours;
  • Step 5 The GelMA with leaf vein structure of step 4 was sterilized by ultraviolet for 30 minutes, placed in a complete medium for vascular endothelial cells and allowed to stand overnight, then inoculated with umbilical vein endothelial cells, and pre-vascularized materials can be obtained after in vitro culture.
  • Vascular endothelial cell complete medium endothelial cell basal medium, 5-10% FBS, 1% double antibody, vascular growth factor; the vascular growth factor includes: vascular endothelial cell growth factor (VEGF), fibroblast growth factor ( FGF), platelet growth factor (PDGF), transforming growth factor ⁇ (TGF ⁇ ) one or more of them.
  • VEGF vascular endothelial cell growth factor
  • FGF fibroblast growth factor
  • PDGF platelet growth factor
  • TGF ⁇ transforming growth factor ⁇
  • a pre-vascularized three-dimensional vascular structure with a hollow structure is obtained.
  • the pre-vascularized material can be used in the preparation of wound repair materials.
  • HUVECs Umbilical Vein Endothelial Cells
  • PBS phosphate buffered saline
  • Red is actin (cytoskeleton protein)
  • blue is DAPI (nuclear marker)
  • white line in Figure 6A is the vein structure
  • scale bar in the lower right corner is 100 ⁇ m.
  • Fig. 7 adopts the same experimental method as Fig. 6A: the dyeing method is the same as Fig. 6A. Then use a confocal laser microscope (Confocal, Leica, confocal laser microscope) to take pictures. Fig. 7 photographs the pre-vascularized material with a three-dimensional vascularized structure with a hollow structure in different parts from a cross-sectional angle. The three-dimensional structure and the cell layer formed on the channel surface can be clearly seen from the figure.
  • a confocal laser microscope Confocal, Leica, confocal laser microscope
  • a method for preparing a biomimetic prevascularized material includes the following steps:
  • Step 1 Vein structure production: Choose leaves with reticulated veins, which are moderately mature, with pale yellow flesh and yellowish brown veins. Put the leaves in water and boil for 5 minutes, wash away the residual solution with running water, scrape gently with tweezers or brush to remove the leaf flesh. After washing, the veins can be obtained, and the whole vein structure is preserved. After washing, use it for later use;
  • vein structure Place the vein structure in a petri dish or other container and moisten it to make it fit completely with the bottom surface of the container; the method of laying veins: the back side of the vein structure faces up, and the other side of the vein structure faces the petri dish;
  • Step 2 PDMS film making: use commercially available Dow Corning SYLGARDTM 184 silicone elastomer and make PDMS according to its instructions. Mix the silicone and curing agent at an 8:1 ratio. After mixing, centrifuge at 3000 rpm/min for 5 Minutes; Pour the centrifuged PDMS into a petri dish to cover the veins in step 1, with a thickness of about 4mm, and place it on the tabletop, vacuum to reduce bubbles; react at 25°C for 24 hours after the PDMS solidifies. After returning to room temperature, remove the veins to obtain a PDMS film with veins "mirror structure";
  • Step 3 PDMS inverted mold: Prepare PDMS according to the method of step 2 again, and spread it on the surface of the PDMS film with the "mirror structure" of the veins in step 2, the covering thickness of PDMS is about 5mm, and the rest is the same as in step 2, and the veins are obtained.
  • Step 4 Place the GelMA solution on the surface of the PDMS template obtained in step 3 for cross-linking and curing, and then remove the PDMS template to obtain GelMA with a vein structure;
  • DPBS solution was used to prepare 5% GelMA, mixed with APS and TEMED, and quickly spread on the surface of the PDMS template obtained in step 3 for cross-linking and curing, and then let it stand at -20°C for 8 hours and reheat at 4°C 12 hours;
  • Step 5 Place the GelMA with leaf vein structure in step 4 in a sterile environment, wash twice with sterile DPBS at 37°C, soak in DPBS for 2 minutes each time, and then place it in stem cell culture medium for 24 hours One hour later, adipose stem cells and vascular endothelial cells were sequentially inoculated, and the prevascularized material was obtained after cell culture in vitro.
  • Stem cell culture medium 10% FBS, ⁇ -MEM, 1% double antibody.
  • a pre-vascularized material with a three-dimensional vascularized structure with a hollow structure is obtained.
  • the pre-vascularized material is used in the preparation of repair stent materials.
  • Adipose stem cells were seeded on a pre-vascularized material with a hollow structure and three-dimensional vascular structure at a cell density of 5 ⁇ 10 4 cells/cm 2 , and then vascular endothelial cells were seeded with a cell density of 40 ⁇ 10 4 cells /cm 2 .
  • PBS phosphate buffered saline
  • Red is actin (cytoskeleton protein), blue is DAPI (nuclear marker), and the scale bar in the lower right corner is 100 ⁇ m.
  • a method for preparing a biomimetic prevascularized material includes the following steps:
  • Step 1 Place the commercially available leaf vein structure in a petri dish or other container and moisten it to make it fit completely on the bottom surface of the container; leaf vein laying method: the back side of the leaf vein structure is facing up, and the other side of the leaf vein structure is facing the culture Dish
  • Step 2 For PDMS membrane production, use commercially available Dow Corning SYLGARDTM 184 silicone elastomer and make PDMS according to its instructions. Mix the silicone and curing agent at 12:1, mix well, and centrifuge at 3000rpm/min for 5 minutes; Pour the centrifuged PDMS into a petri dish to cover the veins in step 1, with a thickness of about 0.1 mm, and place it on the tabletop, vacuum to reduce bubbles; react at 100°C for 8 hours after the PDMS solidifies. After returning to room temperature, remove the veins to obtain a PDMS film with veins "mirror structure";
  • Step 3 PDMS inverted mold: prepare PDMS according to the method of step 2 again, and spread it on the surface of the PDMS film with leaf vein "mirror structure" in step 2, the covering thickness of PDMS is about 1mm, and the rest is the same as in step 2, and the leaf veins are obtained.
  • Step 4 Place the GelMA solution on the surface of the PDMS template obtained in step 3 for cross-linking and curing, and then remove the PDMS template to obtain GelMA with a vein structure;
  • GelMA is added to the Irgacure 2959 solution with a concentration of 1% and mixed, and a certain amount of the mixed solution is poured on the surface of the PDMS template in step S3, irradiated under a UV lamp for 30 seconds, and cross-linked and cured.
  • the cross-linking curing molding conditions are 365nm, 15W UV lamp irradiation for 30 seconds.
  • Step 5 Place the GelMA with leaf vein structure of step 4 in a sterile environment, wash twice with sterile DPBS at room temperature, soak in DPBS for 10 minutes each time, and then place it in the fibroblast culture medium. Six hours later, fibroblasts and vascular endothelial cells were inoculated, and the prevascularized material was obtained after in vitro cell culture. Fibroblast culture medium: DMEM, 10% FBS, 1% double antibody.
  • a pre-vascularized material with a three-dimensional vascularized structure with a hollow structure is obtained.
  • the pre-vascularized material is applied in the preparation of tissue filling/replacement materials or wrapping materials in the field of plastic surgery and beauty.
  • Wound and tissue repair materials, scaffold materials, organ reconstruction materials, tissue replacement materials, tissue filling materials or wrapping materials, in-situ induction regeneration materials include: subcutaneous filling materials, dermal tissue atrophy or defect filling materials, muscle tissue defect filling or replacement materials , Breast filling or replacement material, nasal filling or replacement material, hip filling or replacement material, or prosthesis wrapping material, artificial vascularized skin flap, vascularized artificial tissue, in situ suture replacement material, supplement of missing or inactivated tissue/organ One or more of the materials.
  • Experimental method Inoculate fibroblasts on a pre-vascularized material with a hollow structure and three-dimensional vascular structure at a cell density of 5 ⁇ 10 4 cells/cm 2 , and then inoculate vascular endothelial cells at a density of 40 ⁇ 10 4 cells/cm 2 .
  • PBS phosphate buffered saline
  • Red is actin (cytoskeleton protein), blue is DAPI (nuclear marker), and the scale bar in the lower right corner is 100 ⁇ m.
  • a preparation method of pre-lymphangiized material with bionic vascular structure including the following steps:
  • Step 1 Vein structure production: Choose leaves with reticulated veins, which are moderately mature, with pale yellow flesh and yellowish brown veins. Put the leaves in a mixed solution of 3.5% sodium hydroxide and 2.5% anhydrous sodium carbonate and boil for 5-10 minutes, wash away the corrosive solution in clean water, and then brush off the leaf flesh with a brush. The veins can be obtained after washing Complete vein structure, spare;
  • vein structure Place the vein structure in a petri dish or other container and moisten it to make it fit completely with the bottom surface of the container; the method of laying veins: the back side of the vein structure faces up, and the other side of the vein structure faces the petri dish;
  • Step 2 PDMS film making: use commercially available Dow Corning SYLGARDTM 184 silicone elastomer and make PDMS according to its instructions. Mix the silicone and curing agent at 10:1, mix well and centrifuge at 2000rpm/min for 15 minutes ; Pour the centrifuged PDMS into a petri dish to cover the veins in step 1, with a thickness of about 3mm, and place it on the tabletop, vacuum to reduce bubbles; react at 150°C for 10 minutes after the PDMS solidifies. After returning to room temperature, remove the veins to obtain a PDMS film with veins "mirror structure";
  • Step 3 PDMS inverted mold: Prepare PDMS according to the method of step 2 again, and spread it on the surface of the PDMS film with the "mirror structure" of the veins in the step 2, the covering thickness of PDMS is about 3mm, and the rest is the same as in step 2, and the veins are obtained.
  • Step 4 Place the GelMA solution on the surface of the PDMS template obtained in step 3 for cross-linking and curing, and then remove the PDMS template to obtain GelMA with a vein structure.
  • GelMA is added to the Irgacure 2959 solution with a concentration of 1% and mixed, and a certain amount of the mixed solution is poured on the surface of the PDMS template in step S3, and irradiated under a 365nm, 20W UV lamp for 10 seconds to cross-link and cure to form;
  • Step 5 After the GelMA with vein structure of step 4 is sterilized with ethylene oxide gas, it is placed in a complete medium for lymphatic endothelial cells and then inoculated for 12 hours before inoculation of lymphatic endothelial cells. After cell culture in vitro, the pretreatment is obtained. Lymphatic materials.
  • Lymphatic endothelial cell complete medium endothelial cell basal medium, 5-10% FBS, 1% double antibody, vascular growth factor; the vascular growth factor includes vascular endothelial cell growth factor (VEGF), fibroblast growth factor ( FGF), platelet growth factor (PDGF), transforming growth factor ⁇ (TGF ⁇ ) one or more of them.
  • VEGF vascular endothelial cell growth factor
  • FGF fibroblast growth factor
  • PDGF platelet growth factor
  • TGF ⁇ transforming growth factor ⁇
  • a prelymphatic material with a three-dimensional vascularized structure with a hollow structure is obtained.
  • the pre-lymphatic vascularization material is applied in the preparation of repairing scaffold materials.
  • Lymphatic endothelial cells were seeded on a pre-lymphangiized material with a hollow three-dimensional vascularized structure, and the cell density was 40 ⁇ 10 4 cells/cm 2 .
  • PBS phosphate buffered saline
  • Red is actin (cytoskeleton protein), blue is DAPI (nuclear marker), and the scale bar in the lower right corner is 100 ⁇ m.
  • Fig. 10 it can be seen from Fig. 10 that in the bionic pre-lymphatic material, the cells grow distributed along the vein structure, and the reticular branch structure with leaf veins formed is similar to the lymphatic network structure of the human body.
  • a method for preparing a biomimetic prevascularized material includes the following steps:
  • Steps 1 to 3 of Examples 5 to 7 are all the same as Example 1.
  • Step 4 2%-20% by mass volume ratio of gelatin (from porcine or cattle) was dissolved in deionized water, and spread on the surface of the PDMS template obtained in step 3, with a thickness of 0.1 ⁇ 2mm, with 0.25% ⁇ 2.5% glutaraldehyde solution is cross-linked at 37°C for 4-6 hours.
  • the crosslinked gelatin material is soaked and washed with deionized water to remove unreacted glutaraldehyde. Then remove the PDMS template to obtain gelatin with leaf vein structure;
  • Step 4 2%-20% by mass volume ratio of gelatin (from porcine or cattle) was dissolved in deionized water and spread on the surface of the PDMS template obtained in step 3 with a thickness of 0.1-2mm and 0.5-2g /L EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was crosslinked at 37°C for 4-8 hours. The crosslinked gelatin material is soaked and washed with deionized water to remove unreacted glutaraldehyde. Then remove the PDMS template to obtain gelatin with leaf vein structure;
  • Step 4 2%-20% by mass volume ratio of gelatin (from porcine or cattle) was dissolved in deionized water and spread on the surface of the PDMS template obtained in step 3, with a thickness of 0.1-2mm, using EDC/NHS Crosslinking, 0.5-2g/L EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 0.1-0.5g/L NHS (N-hydroxysuccinimide) is cross-linked at 37°C for 4-8 hours. The crosslinked gelatin material is soaked and washed with deionized water, EDC and NHS. Then remove the PDMS template to obtain gelatin with leaf vein structure;
  • Step 5 Place the leaf-veined gelatin in step 4 in a sterile environment, wash twice with sterile DPBS at room temperature, soak in DPBS for 10 minutes each time, and then place it in induced pluripotent stem cells (iPS cells). ) After being allowed to stand for 6 hours in the culture medium, induced pluripotent stem cells (iPS cells) are seeded, and vascular endothelial cells are multiple seeded, and the prevascularized material is obtained after cell culture in vitro.
  • Stem cell culture medium Essential 6TM Medium, vascular endothelial cell culture medium as described above.
  • a prevascularized material with a three-dimensional vascularized structure with a hollow structure is obtained.
  • the prevascularized material is used in the preparation of wound repair materials.
  • iPS cells Induced pluripotent stem cells
  • vascular endothelial cells were seeded on a pre-vascularized material with a hollow three-dimensional vascularized structure, and the cell density was 5 ⁇ 10 4 cells/cm 2 and 40 ⁇ 10, respectively 4 cells/cm 2 .
  • PBS phosphate buffered saline
  • Red is actin (cytoskeleton protein), blue is DAPI (nuclear marker), and the scale bar in the lower right corner is 100 ⁇ m.
  • the cells grow distributed along the vein structure, and the formed reticular branch structure with leaf veins is similar to the microcirculation network structure of the human body, which can be at least early in wound repair Establish blood circulation in 3 days and improve the survival rate of the graft.
  • a method for preparing a biomimetic prevascularized material includes the following steps:
  • Steps 1 to 4 of Example 8 are the same as those of Example 1.
  • Step 5 of Example 5 After superimposing GelMA with mirror-mirror structure after sterilization with ethylene oxide gas, cells (microvascular endothelial cells) were perfused and cultured to obtain a pre-vessel with a hollow structure of three-dimensional vascularized network structure ⁇ Material.
  • the GelMA with leaf vein structure prepared in Example 1 was inoculated with cells (umbilical vein endothelial cells, HUVECs), superimposed, and transplanted into nude mice. After 3 weeks, blood vessels grew into the gel, as indicated by the black arrow in Figure 12 For blood vessels or vascular network.
  • Figures 12A and 12B are the roots of the blood vessels
  • Figure 12C is the end of the blood vessels
  • Figure 13 is the 12th week after implantation in the body. It can be seen that the blood vessels of the nude mice grow into the vein structure, and their course is similar to the vein structure.

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Abstract

一种仿生的预脉管化材料及其制备方法和应用。所述预脉管化材料的制备方法包括制备具有叶脉结构的生物材料，利用所述的具有叶脉结构的生物材料进行细胞培养等步骤。所述预脉管化材料可用于制备创面修复材料、支架材料、器官再造材料、组织替代材料、组织填充材料或包裹材料等，特别的可用于制备具有与人体淋巴管网结构或微血管结构相似的预淋巴管化材料和预血管化材料。

Description

一种仿生的预脉管化材料及其制备方法和应用 技术领域
本发明涉及生物医药的技术领域，更具体的，涉及一种仿生的预脉管化材料及其制备方法和应用。
 背景技术
血管化是创面修复的必要条件。早期血管化可以提高移植物成活率，创面修复效率和质量。现已证明预血管化的材料较未预血管化的材料可提早3天建立血液循环，可见，预血管化材料的制备和应用具有很强的必要性和良好的应用效果。
各种原因引起淋巴管损伤和功能障碍所导致的淋巴水肿和组织纤维化，严重影响患者的生活质量。但是修复淋巴管损伤，改善淋巴管功能的方法和手段仍未有明显改进。因此，探索一种可以修复淋巴管功能和结构的方式具有其必要性和重要性。
既往的研究中，使用的预血管化材料和制备手段主要有：使用支架材料，如脱细胞基质，复合种子细胞（干细胞、血管内皮细胞）等体外培养获得预血管化材料；或者使用细胞膜片技术，如Okano教授等人利用温敏材料体外培养细胞膜片，复合血管内皮细胞后获得具有3D结构的预血管化材料，现已应用于角膜、食管等组织器官的修复研究。预淋巴管化材料的制备手段主要是皮肤组织中提取的混合细胞，体外培养获得淋巴管和血管的材料的技术。
以上技术的总特征为利用生物、化学、材料学或工程技术手段制备支架材料，复合血管内皮细胞构建血管化材料。其中生物技术方法需要选择合适的异体或异种的组织，使用化学或物理的方法脱细胞，以获得基质材料，整个流程相对复杂，耗时较长，在临床应用方面更是涉及伦理和排异问题；体外培养细胞膜片，需要具有制备温敏材料的技术和平台，体外培养具有足够组织量的3D血管化材料需要多层膜片叠加，存在技术手段相对复杂，操作流程较多的问题；高分子材料涉及材料的改性、微观结构、组织形容性、降解率及其降解产物毒性等问题。皮肤组织混合细胞培养的方式，细胞提取和培养周期较长，获得的组织厚度不足，不适用于大面积的皮肤或组织修复等。
 技术问题
本发明解决的技术问题是克服现有的问题，提供一种仿生的预脉管化材料；
本发明的另一目的在于提供一种仿生的预脉管化材料的制备方法；
本发明的另一目的在于提供一种仿生的预脉管化材料在制备创面和组织修复材料、支架材料、器官再造材料、组织填充材料或包裹材料中的应用。
 技术解决方案
本发明通过以下技术方案予以实现：
一种仿生的预脉管化材料的制备方法，包括以下步骤：
步骤1. 制备具有叶脉结构的生物材料；
步骤2. 将步骤1中获得的生物材料消毒、清洗后，置于细胞培养基中静置过夜，然后接种细胞进行体外细胞培养；
将接种有细胞的互成镜像结构的生物材料叠加，得到具有中空结构的三维脉管化网络结构的预脉管化材料；
或，
将接种有细胞的具有叶脉结构的生物材料与无叶脉结构的生物材料叠加后，得到具有中空结构的三维脉管化网络结构的预脉管化材料；
或，
将消毒后的互成镜像结构的生物材料叠加，或者将消毒后的具有叶脉结构的生物材料与无叶脉结构的生物材料叠加后进行细胞灌注培养，得到具有中空结构的三维脉管化网络结构的预脉管化材料。
优选地，所述生物材料包括但不限于：（1）天然多糖，如纤维素、甲壳素、壳聚糖、香菇多糖；（2）两性多糖衍生物，如聚乳酸、聚已内酯及其衍生物；（3）天然高分子类水凝胶，如壳聚糖水凝胶；（4）GelMA、明胶、透明质酸及其改良材料等。
优选地，步骤1.包括以下步骤：
步骤11. 将天然植物的叶脉结构置于培养皿或其他容器中，背面朝上，润湿，使其与容器底面贴合；
步骤12. 聚二甲基硅氧烷制膜：制备PDMS并将其覆盖步骤1中叶脉结构表面进行固化后，取下叶脉后得到具有叶脉“镜面结构”的PDMS薄膜；
步骤13. 聚二甲基硅氧烷倒模：制备PDMS并将其平铺于步骤2中的具有叶脉“镜面结构”的PDMS薄膜的表面进行固化后，取下具有叶脉“镜面结构”的PDMS薄膜，得到与叶脉相同结构的PDMS模板；
步骤14. 取GelMA溶液或明胶置于步骤3得到的PDMS模板的表面进行交联固化成型，然后取下PDMS模板，得具有叶脉结构的GelMA或明胶。
优选地，步骤1包括以下步骤：
通过3D打印技术，以生物材料为“打印墨水”，以叶脉结构为模板，打印出具有叶脉结构的生物材料。
优选地，步骤1包括以下步骤：
以激光打印技术为基础，以固化的生物材料为基质，以叶脉结构为模板，打印出具有叶脉结构的生物材料。
所述预脉管化包括预血管化和预淋巴管化。
本发明公开了一种利用仿生技术制备预脉管化组织工程材料，其由生物相容的可吸收性支架及由一种或多种细胞，并且采用或未采用生物反应器、微流控设备处理和培养。此类预脉管化材料可用于修复或填充受损或病变组织。在组织工程材料领域，脉管化或血管化的速度和效率影响移植物的存活率及其组织填充和修复效果。预脉管化的材料可以明显缩短这一过程。因此本发明中使用的仿生技术，即利用叶脉走形与组织微循环脉管化结构相似这一特点，使用“拓模、3D打印技术、激光打印技术等”方法，使用具有生物相容性的生物材料（包括但不限于GelMA或明胶等）与细胞培养技术，获得预脉管化材料。
本发明中具有叶脉结构表面的GelMA或明胶起到支架材料的功能，可以根据需要对其进行改进，使其具有适于可预期的应用机械性能，可保持足够的完整性直至组织向内生长并愈合；具有易于加工塑形为多种产品的特性和可接受的储存寿命；但不会在体内引起明显的炎性反应或毒性反应。
优选地，步骤11中所述天然植物的叶脉结构由以下任一方法制备得到：
方法一：将树叶置于8~12%氢氧化钠溶液中煮沸5~10分钟后，流水洗去残留氢氧化钠溶液，镊子轻刮或毛刷刷去叶肉，清洗后可获得叶脉；
方法二：将树叶放在水中煮沸5~10分钟后，流水洗去残留溶液，镊子轻刮或毛刷刷去叶肉，清洗后可获得叶脉；
方法三：将树叶置于3~5%氢氧化钠和2~5%无水碳酸钠的混合溶液中煮沸5~10分钟后，在清水中洗去腐蚀溶液，再用毛刷刷掉叶肉，清洗后可获得叶脉。
优选地，所述制备PDMS的方法为：将硅酮和固化剂混合，两者混合比例8~12:1，除气泡后，于温度25~150℃下，固化反应10分钟~24小时。进一步优选地，PDMS固化条件为：于温度65℃下固化反应4小时。
优选地，步骤12和步骤13中PDMS薄膜的厚度为1mm~10mm；进一步优选地，步骤12和步骤13中PDMS薄膜的厚度为2mm~5mm；
优选地，除气泡的方法包括抽真空、离心。所述离心方法：将硅酮和固化剂混匀后以1500~3000rpm/分，离心5~30分钟。
优选地，步骤14中所述交联固化包括以下任一方法：
用磷酸盐缓冲液(DPBS)制备浓度5%~20% GelMA，与四甲基乙二胺(TEMED)混合，加入冰上预冷的过硫酸铵，迅速混匀后，即平铺于步骤3得到的已于冰上或2~8℃预冷的PDMS模板的表面进行交联固化成型。其中优选地，固化成型条件为于-20℃静置8~24小时，然后于4℃复温4~24小时。
或，在浓度为0.01%~1%的Irgacure2959溶液中加入GelMA混合，取一定量混合液倒于由步骤S13中的PDMS模板表面，在UV灯下照射10~600秒，交联固化成型。其中优选地，交联固化成型条件为在365nm，10~20W的UV灯下照射10~60秒。
优选地，步骤2中的消毒方法包括以下任一方法：
具叶脉结构的GelMA或明胶置于在无菌环境中，室温下置于70%~75%酒精中浸泡30分钟，再使用无菌磷酸盐缓冲液（DPBS）或PBS清洗1~5次，每次浸泡2~10分钟；
或，对具叶脉结构的GelMA或明胶进行紫外光消毒或气体消毒。优选地，紫外光消毒30分钟；所述气体消毒为采用环氧乙烷气体消毒。
或，对具叶脉结构的GelMA或明胶进行70%~75%酒精浸泡同时紫外消毒或气体消毒。气体消毒时间为30分钟。
优选地，步骤2中所述细胞和细胞培养基包括以下的一种或几种：
1）血管内皮细胞，包括自体、异体或异种来源的脐静脉血管内皮细胞、微血管内皮细胞、内皮祖细胞；
血管内皮细胞完全培养基：内皮细胞基础培养基，5~10%FBS，1%双抗，血管生长因子；所述血管生长因子包括促进内皮细胞分裂增殖和/或分化的因子或蛋白；
2）干细胞，所述干细胞包括，自体、异体或异种来源的胚胎干细胞和成体干细胞，脂肪干细胞、骨髓间充质干细胞、表皮干细胞、神经干细胞；
干细胞培养基：10%FBS，α-MEM、间充质干细胞培养基（MSCM）、高糖培养基（DMEM）和/或定向分化培养基，1%双抗；
3）成纤维细胞；自体、异体或异种来源的成纤维细胞培养基：DMEM，10%FBS，1%双抗；
4）自体、异体或异种来源的诱导性多能干细胞（iPS细胞），干细胞培养基：Essential 6™ Medium；
5) 自体、异体或异种来源的淋巴管内皮细胞；淋巴管内皮细胞完全培养基：内皮细胞基础培养基，5-10%FBS，1%双抗，血管生长因子；所述血管生长因子包括促进内皮细胞分裂增殖和分化的因子或蛋白。
优选地，所述脐静脉血管内皮细胞是异体的，微血管内皮细胞、内皮祖细胞可是自体、异体或异种来源的。
优选地，所述血管生长因子包括：血管内皮细胞生长因子（VEGF）、成纤维细胞生长因子（FGF）、血小板生长因子（PDGF）、转化生长因子β（TGFβ）中的一种或几种，促进内皮细胞分裂增殖和/或分化的因子或蛋白。
一种由上述制备方法制备得到的仿生的预脉管化材料。
一种上述仿生的预脉管化材料在制备创面和组织修复材料、支架材料、器官再造材料、组织替代材料、组织填充材料或包裹材料、原位诱导再生材料中的应用。
本发明中构建的预脉管化材料，既可以应用实体组织或器官的修复，如皮肤、乳腺、肝脏、脾脏；也可以通过改良支架材料的物理特性制备成修复食管、气管、胃、肠道、心脏等空腔脏器的内膜或外层修复；还可以用于整形美容领域中的组织填充或包裹等。
优选地，所述仿生的预脉管化材料在接种细胞后可叠加，或与无叶脉结构表面的预脉管化材料叠加，形成具有中空结构的三维脉管化网络结构。
优选地，优选地，所述创面和组织修复材料、支架材料、器官再造材料、组织替代材料、组织填充材料或包裹材料、原位诱导再生材料包括：皮下填充材料、真皮组织萎缩或缺损填充材料、肌组织缺损填充或替代材料、乳房填充或替代材料、鼻填充或替代材料、臀部填充或替代材料、或假体包裹材料、人造血管化皮瓣、血管化人造组织、原位缝合替代材料、补充缺失或失活组织/器官的材料中的一种或几种。
 有益效果
与现有技术相比，本实用新型的有益效果：
本发明制备得到的仿生的预脉管化材料，具有树叶叶脉的网状分支结构与人体的微循环网络结构相似，在创面修复中能够至少提早3天建立血液循环，提高移植物成活率。
本发明中所使用的天然植物的叶脉结构，广泛存在，易于获得，可根据不同的器官和组织中对脉管直径的需要选择不同树种来源的叶子作为结构模板。
本发明中使用的仿生技术，创新地利用树叶的叶脉走形与组织微循环脉管化结构相似这一特点，使用“拓模”的方法，制备出与叶脉相同结构的PDMS模板。
本发明中具有叶脉结构表面的生物材料（包括但不限于GelMA或明胶等）起到支架材料的功能，可以根据需要对其进行改进，调整交联方案，使其具有适于可预期的应用机械性能，可保持足够的完整性直至组织向内生长并愈合；具有易于加工塑形为多种产品的特性和可接受的储存寿命；但不会在体内引起明显的炎症反应或毒性反应。
 附图说明
图1为天然植物的叶脉结构；
图2为具有叶脉“镜面结构”的聚二甲基硅氧烷薄膜；
图3为具有叶脉结构的聚二甲基硅氧烷模板；
图4为叶脉结构表面GelMA；
图5中的A图为GelMA，B图为叶脉结构表面的GelMA；
图6中的A图在具有叶脉结构表面的GelMA表面接种血管内皮细胞后，免疫荧光染色图；B图为无叶脉结构表面的GelMA表面接种血管内皮细胞后，免疫荧光染色图，红色为actin，蓝色为DAPI。
图7为采用激光共聚焦显微镜（Confocal， Leica，激光共聚焦显微镜）拍摄的照片。
图8~11分别为实施例2~5的免疫荧光染色图，红色为actin，蓝色为DAPI，以及红色和蓝色的叠加图。
图12为具有中空结构的三维脉管化结构的预血管化材料植入裸鼠体内3周后，取出拍摄的图片。
图13为具有中空结构的三维脉管化结构的预血管化材料植入裸鼠体内12周的图片。
 本发明的最佳实施方式
本发明可以结合以下具体实施例进一步解释和阐明，但具体实施例并不对本发明有任何形式的限定。
以下实施例中使用的原材料、设备、化学品、培养基等均为普通市购获得。
实施例 1
一种仿生的预血管化材料的制备方法，包括以下步骤：
步骤1. 叶脉结构制作：选择具有网状叶脉的树叶，中度成熟，叶肉淡黄色，叶脉黄褐色。将树叶置于10%氢氧化钠溶液中煮沸10分钟后，流水洗去残留氢氧化钠溶液，镊子轻刮或毛刷刷去叶肉，保留完整叶脉结构，如图1所示；
将叶脉结构置于培养皿或其他容器中，润湿，使其完全与容器底面贴合；叶脉铺置方法：叶脉结构的背面朝上（即树叶背面朝上），叶脉结构的另一面朝向培养皿；
步骤2. PDMS制膜：使用市购来的道康宁SYLGARD™184有机硅弹性体并按其说明书制作PDMS，将硅酮和固化剂以10:1混合，混匀后以1500rpm/分，离心30分钟；将离心后的PDMS倒于培养皿中覆盖步骤1中叶脉结构表面，厚度约2mm，静置于桌面，抽真空，减少气泡；65℃反应4小时PDMS固化后。恢复至室温后，取下叶脉后得到具有叶脉“镜面结构”的PDMS薄膜，如图2所示；
步骤3. PDMS倒模：再次按步骤2的方法制备PDMS，平铺于步骤2中的具有叶脉“镜面结构”的PDMS薄膜的表面，PDMS的覆盖厚度约2mm，其余同步骤2，得到与叶脉相同结构的PDMS模板，如图3所示；
步骤4.取GelMA溶液置于步骤3得到的PDMS模板的表面进行交联固化成型，然后取下PDMS模板，得具有叶脉结构的GelMA，如图4和5所示；
具体的，用DPBS溶液制备浓度5%GelMA，与APS和TEMED混合后迅速平铺于步骤3得到中的PDMS模板的表面进行交联固化成型，于-20℃静置12小时，4℃复温24小时；
步骤5.将步骤4的具叶脉结构的GelMA紫外消毒30分钟，放入血管内皮细胞完全培养基中静置过夜后，接种脐静脉血管内皮细胞，体外培养后可以获得预血管化材料。
血管内皮细胞完全培养基：内皮细胞基础培养基，5-10%FBS，1%双抗，血管生长因子；所述血管生长因子包括：血管内皮细胞生长因子（VEGF），成纤维细胞生长因子（FGF），血小板生长因子（PDGF），转化生长因子β（TGFβ）中的一种或几种。
将接种有细胞的具有叶脉结构的GelMA（图7中标示“leaf”）与无叶脉结构的GelMA（图7中标示“flat”）叠加后，得到具有中空结构的三维脉管化结构的预血管化材料，该预血管化材料可在制备创面修复材料中应用。
预血管化材料的表征实验：
实验方法：将HUVECs（脐静脉血管内皮细胞）接种于具有中空结构的三维脉管化结构的预血管化材料，细胞密度为40×10 4 cells/cm 2。第3天，吸取培养基，用PBS（磷酸盐缓冲液）洗2次，5分钟每次，加入10%中性多聚甲醛溶液，室温固定30分钟，吸去甲醛后，加入PBS 洗3次。加入0.2%Triton-PBS，室温30分钟，吸除，再加入1%BSA-PBS，室温封闭1小时，洗净，加入Actin（1:50）溶液，孵育1小时，吸去，PBS洗3次，5分钟/次，加入DAPI (1:1000) ，孵育8分钟。PBS洗3次，5分钟/次。PBS少量，保持湿润，荧光显微镜（Leica DMi8）下拍照，如图6A所示。
说明：红色为actin（细胞骨架蛋白），蓝色为DAPI（细胞核标记物），图6A中白色线为叶脉结构，右下角比例尺为100µm。
图7采用与图6A相同的实验方法：即染色方法与图6A相同。然后采用激光共聚焦显微镜（Confocal， Leica，激光共聚焦显微镜）拍照。图7从截面角度拍摄不同部位具有中空结构的三维脉管化结构的预血管化材料，从图中可以明显看出三维结构，以及在通道表面形成的细胞层。
从图6A可以看出在该仿生脉管结构的预血管化材料，细胞沿着叶脉结构进行分布生长，形成的具有树叶叶脉的网状分支结构与人体的微循环网络结构相似，在创面修复中能够至少提早3天建立血液循环，提高移植物成活率。相比较图6B可知，无叶脉结构表面的GelMA表面接种血管内皮细胞后，无法形成与人体的微循环网络结构相似的预血管化材料。
实施例 2
一种仿生的预血管化材料的制备方法，包括以下步骤：
步骤1. 叶脉结构制作：选择具有网状叶脉的树叶，中度成熟，叶肉淡黄色，叶脉黄褐色。将树叶放在水中煮沸5分钟后，流水洗去残留溶液，镊子轻刮或毛刷刷去叶肉，清洗后可获得叶脉，保留完整叶脉结构，清洗后备用；
将叶脉结构置于培养皿或其他容器中，润湿，使其完全与容器底面贴合；叶脉铺置方法：叶脉结构的背面朝上，叶脉结构的另一面朝向培养皿；
步骤2. PDMS制膜：使用市购来的道康宁SYLGARD™184有机硅弹性体并按其说明书制作PDMS，将硅酮和固化剂以8:1混合，，混匀后以3000rpm/分，离心5分钟；将离心后的PDMS倒于培养皿中覆盖步骤1中叶脉，厚度约4mm，静置于桌面，抽真空，减少气泡；25℃反应24小时PDMS固化后。恢复至室温后，取下叶脉后得到具有叶脉“镜面结构”的PDMS薄膜；
步骤3. PDMS倒模：再次按步骤2的方法制备PDMS，平铺于步骤2中的具有叶脉“镜面结构”的PDMS薄膜的表面，PDMS的覆盖厚度约5mm，其余同步骤2，得到与叶脉相同结构的PDMS模板；
步骤4. 取GelMA溶液置于步骤3得到的PDMS模板的表面进行交联固化成型，然后取下PDMS模板，得具有叶脉结构的GelMA；
具体的，用DPBS溶液制备浓度5%GelMA，与APS和TEMED混合后迅速平铺于步骤3得到中的PDMS模板的表面进行交联固化成型，于-20℃静置8小时，4℃复温12小时；
步骤5. 将步骤4的具叶脉结构的GelMA置于在无菌环境中，37℃下使用无菌DPBS清洗2次，每次在DPBS中浸泡2分钟，然后置于干细胞培养基中静置24小时后依次接种脂肪干细胞和血管内皮细胞，体外细胞培养后获得所述预血管化材料。干细胞培养基：10%FBS，α-MEM，1%双抗。
将接种有细胞的具有叶脉结构的GelMA与无叶脉结构表面的GelMA叠加后，得到具有中空结构的三维脉管化结构的预血管化材料。将该预血管化材料在制备修复支架材料中应用。
预血管化材料的表征实验：
实验方法：将脂肪干细胞接种于具有中空结构的三维脉管化结构的预血管化材料，细胞密度为5×10 4 cells/cm 2，后再接种血管内皮细胞，细胞密度为40×10 4 cells/cm 2。第3天，吸取培养基，用PBS（磷酸盐缓冲液）洗2次，5分钟每次，加入10%中性甲醛溶液，室温固定30分钟，吸去甲醛后，加入PBS 洗3次。加入0.2%Triton-PBS，室温30分钟，吸除，再加入1%BSA-PBS，室温封闭1小时，洗净，加入Actin（1:50）溶液，孵育1小时，吸去，PBS洗3次，5分钟/次，加入DAPI (1:1000) ，孵育8分钟。PBS洗3次，5分钟/次。PBS少量，保持湿润，荧光显微镜（Leica DMi8）下拍照，如图8所示。
说明：红色为actin（细胞骨架蛋白），蓝色为DAPI（细胞核标记物），右下角比例尺为100µm。
从图8可以看出在该仿生的预血管化材料，细胞沿着叶脉结构进行分布生长，形成的具有树叶叶脉的网状分支结构与人体的微循环网络结构相似，在创面修复中能够至少提早3天建立血液循环，提高移植物成活率。
实施例 3
一种仿生的预血管化材料的制备方法，包括以下步骤：
步骤1.将市购来的叶脉结构置于培养皿或其他容器中，润湿，使其完全与容器底面贴合；叶脉铺置方法：叶脉结构的背面朝上，叶脉结构的另一面朝向培养皿；
步骤2. PDMS制膜使用市购来的道康宁SYLGARD™184有机硅弹性体并按其说明书制作PDMS，将硅酮和固化剂以12:1混合，混匀后以3000rpm/分，离心5分钟；将离心后的PDMS倒于培养皿中覆盖步骤1中叶脉，厚度约0.1mm，静置于桌面，抽真空，减少气泡；100℃反应8小时PDMS固化后。恢复至室温后，取下叶脉后得到具有叶脉“镜面结构”的PDMS薄膜；
步骤3. PDMS倒模：再次按步骤2的方法制备PDMS，平铺于步骤2中的具有叶脉“镜面结构”的PDMS薄膜的表面，PDMS的覆盖厚度约1mm，其余同步骤2，得到与叶脉相同结构的PDMS模板；
步骤4. 取GelMA溶液置于步骤3得到的PDMS模板的表面进行交联固化成型，然后取下PDMS模板，得具有叶脉结构的GelMA；
具体的，在浓度为1%的Irgacure2959溶液中加入GelMA混合，取一定量混合液倒于由步骤S3中的PDMS模板表面，在UV灯下照射30秒，交联固化成型。交联固化成型条件为在365nm，15W的UV灯下照射30秒。
步骤5. 将步骤4的具叶脉结构的GelMA置于在无菌环境中，室温下使用无菌DPBS清洗2次，每次在DPBS中浸泡10分钟，然后置于成纤维细胞培养基中静置6小时后接种成纤维细胞和血管内皮细胞，体外细胞培养后获得所述预血管化材料。成纤维细胞培养基：DMEM，10%FBS，1%双抗。
将接种有细胞的具有叶脉结构的GelMA与无叶脉结构表面的GelMA叠加后，得到具有中空结构的三维脉管化结构的预血管化材料。将该预血管化材料在制备整形美容领域的组织填充/替代材料或包裹材料中应用。创面和组织修复材料、支架材料、器官再造材料、组织替代材料、组织填充材料或包裹材料、原位诱导再生材料包括：皮下填充材料、真皮组织萎缩或缺损填充材料、肌组织缺损填充或替代材料、乳房填充或替代材料、鼻填充或替代材料、臀部填充或替代材料、或假体包裹材料、人造血管化皮瓣、血管化人造组织、原位缝合替代材料、补充缺失或失活组织/器官的材料中的一种或几种。
预血管化材料的表征实验：
实验方法：将成纤维细胞接种于具有中空结构的三维脉管化结构的预血管化材料，细胞密度为5×10 4 cells/cm 2，再接种血管内皮细胞，密度为40×10 4 cells/cm 2。第3天，吸取培养基，用PBS（磷酸盐缓冲液）洗2次，5分钟每次，加入10%中性甲醛溶液，室温固定30分钟，吸去甲醛后，加入PBS 洗3次。加入0.2%Triton-PBS，室温30分钟，吸除，再加入1%BSA-PBS，室温封闭1小时，洗净，加入Actin（1:50）溶液，孵育1小时，吸去，PBS洗3次，5分钟/次，加入DAPI (1:1000) ，孵育8分钟。PBS洗3次，5分钟/次。PBS少量，保持湿润，荧光显微镜（Leica DMi8）下拍照，如图9所示。
说明：红色为actin（细胞骨架蛋白），蓝色为DAPI（细胞核标记物），右下角比例尺为100µm。
从图9可以看出在该仿生脉管结构的预血管化材料，细胞沿着叶脉结构进行分布生长，形成的具有树叶叶脉的网状分支结构与人体的微循环网络结构相似，在创面修复中能够至少提早3天建立血液循环，提高移植物成活率。
实施例 4
一种仿生脉管结构的预淋巴管化材料的制备方法，包括以下步骤：
步骤1. 叶脉结构制作：选择具有网状叶脉的树叶，中度成熟，叶肉淡黄色，叶脉黄褐色。将树叶置于3.5%氢氧化钠和2.5%无水碳酸钠的混合溶液中煮沸5-10分钟后，在清水中洗去腐蚀溶液，再用毛刷刷掉叶肉，清洗后可获得叶脉，保留完整叶脉结构，备用；
将叶脉结构置于培养皿或其他容器中，润湿，使其完全与容器底面贴合；叶脉铺置方法：叶脉结构的背面朝上，叶脉结构的另一面朝向培养皿；
步骤2. PDMS制膜：使用市购来的道康宁SYLGARD™184有机硅弹性体并按其说明书制作PDMS，将硅酮和固化剂以10:1混合，混匀后以2000rpm/分，离心15分钟；将离心后的PDMS倒于培养皿中覆盖步骤1中叶脉，厚度约3mm，静置于桌面，抽真空，减少气泡；150℃反应10分钟PDMS固化后。恢复至室温后，取下叶脉后得到具有叶脉“镜面结构”的PDMS薄膜；
步骤3. PDMS倒模：再次按步骤2的方法制备PDMS，平铺于步骤2中的具有叶脉“镜面结构”的PDMS薄膜的表面，PDMS的覆盖厚度约3mm，其余同步骤2，得到与叶脉相同结构的PDMS模板；
步骤4. 取GelMA溶液置于步骤3得到的PDMS模板的表面进行交联固化成型，然后取下PDMS模板，得具有叶脉结构的GelMA。
具体的，在浓度为1%的Irgacure2959溶液中加入GelMA混合，取一定量混合液倒于由步骤S3中的PDMS模板表面，在365nm，20W的UV灯下照射10秒，交联固化成型；
步骤5. 将步骤4的具叶脉结构的GelMA采用环氧乙烷气体消毒后，放入淋巴管内皮细胞完全培养基中静置12小时后接种淋巴管内皮细胞，体外细胞培养后获得所述预淋巴管化材料。
淋巴管内皮细胞完全培养基：内皮细胞基础培养基，5-10%FBS，1%双抗，血管生长因子；所述血管生长因子包括血管内皮细胞生长因子（VEGF），成纤维细胞生长因子（FGF），血小板生长因子（PDGF），转化生长因子β（TGFβ）中的一种或几种。
将接种有细胞的具有叶脉结构的GelMA与无叶脉结构表面的GelMA叠加后，得到具有中空结构的三维脉管化结构的预淋巴管化材料。将该预淋巴管化材料在制备修复支架材料中应用。
预淋巴管化材料的表征实验：
实验方法：将淋巴管内皮细胞接种于具有中空结构的三维脉管化结构的预淋巴管化材料，细胞密度为40×10 4 cells/cm 2。第3天，吸取培养基，用PBS（磷酸盐缓冲液）洗2次，5分钟每次，加入10%中性甲醛溶液，室温固定30分钟，吸去甲醛后，加入PBS 洗3次。加入0.2%Triton-PBS，室温30分钟，吸除，再加入1%BSA-PBS，室温封闭1小时，洗净，加入Actin（1:50）溶液，孵育1小时，吸去，PBS洗3次，5分钟/次，加入DAPI (1:1000) ，孵育8分钟。PBS洗3次，5分钟/次。PBS少量，保持湿润，荧光显微镜（Leica DMi8）下拍照，如图10所示。
说明：红色为actin（细胞骨架蛋白），蓝色为DAPI（细胞核标记物），右下角比例尺为100µm。
从图10可以看出在该仿生的预淋巴管化材料，细胞沿着叶脉结构进行分布生长，形成的具有树叶叶脉的网状分支结构与人体的淋巴管网络结构相似。
实施例 5~7
一种仿生的预血管化材料的制备方法，包括以下步骤：
实施例5~7的步骤1~3均与实施例1相同。
实施例5的步骤4. 2%-20%质量体积比的明胶（猪或牛来源）溶于去离子水中，平铺于步骤3得到的PDMS模板的表面，厚度0.1~2mm，用0.25%~2.5%戊二醛溶液在37℃下交联4~6小时。交联后的明胶材料用去离子水浸泡和清洗，去除未反应的戊二醛。然后取下PDMS模板，得具有叶脉结构的明胶；
实施例6的步骤4. 2%-20%质量体积比的明胶（猪或牛来源）溶于去离子水中，平铺于步骤3得到的PDMS模板的表面，厚度0.1-2mm，用0.5-2g/L EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)在37℃下交联4-8小时。交联后的明胶材料用去离子水浸泡和清洗，去除未反应的戊二醛。然后取下PDMS模板，得具有叶脉结构的明胶；
实施例7的步骤4. 2%-20%质量体积比的明胶（猪或牛来源）溶于去离子水中，平铺于步骤3得到的PDMS模板的表面，厚度0.1-2mm，用EDC/NHS交联，0.5-2g/L EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和0.1-0.5g/L NHS(N-羟基琥珀酰亚胺)在37℃下交联4-8小时。交联后的明胶材料用去离子水浸泡和清洗，EDC和NHS。然后取下PDMS模板，得具有叶脉结构的明胶；
步骤5. 将步骤4的具叶脉结构的明胶置于在无菌环境中，室温下使用无菌DPBS清洗2次，每次在DPBS中浸泡10分钟，然后置于诱导性多能干细胞（iPS细胞）培养基中静置6小时后接种诱导性多能干细胞（iPS细胞），复种血管内皮细胞，体外细胞培养后获得所述预血管化材料。干细胞培养基：Essential 6™ Medium，血管内皮细胞培养基如前所述。
将接种有细胞的具有叶脉结构的明胶叠加后，得到具有中空结构的三维脉管化结构的预血管化材料。将该预血管化材料在制备创面修复材料中应用。
关于实施例 5 制备的预血管化材料的表征实验：
实验方法：将诱导性多能干细胞（iPS细胞）和血管内皮细胞接种于具有中空结构的三维脉管化结构的预血管化材料，细胞密度分别为5×10 4 cells/cm 2和40×10 4 cells/cm 2。第3天，吸取培养基，用PBS（磷酸盐缓冲液）洗2次，5分钟每次，加入10%中性甲醛溶液，室温固定30分钟，吸去甲醛后，加入PBS 洗3次。加入0.2%Triton-PBS，室温30分钟，吸除，再加入1%BSA-PBS，室温封闭1小时，洗净，加入Actin（1:50）溶液，孵育1小时，吸去，PBS洗3次，5分钟/次，加入DAPI (1:1000) ，孵育8分钟。PBS洗3次，5分钟/次。PBS少量，保持湿润，荧光显微镜（Leica DMi8）下拍照，如图11所示。
说明：红色为actin（细胞骨架蛋白），蓝色为DAPI（细胞核标记物），右下角比例尺为100µm。
从图11可以看出在该仿生的预血管化材料，细胞沿着叶脉结构进行分布生长，形成的具有树叶叶脉的网状分支结构与人体的微循环网络结构相似，在创面修复中能够至少提早3天建立血液循环，提高移植物成活率。
实施例 8
一种仿生的预血管化材料的制备方法，包括以下步骤：
实施例8的步骤1~4均与实施例1相同。
实施例5的步骤5. 将采用环氧乙烷气体消毒后的互成镜像结构的GelMA叠加后进行细胞（微血管内皮细胞）灌注培养，得到具有中空结构的三维脉管化网络结构的预脉管化材料。
实施例 9 应用实施例
将实施例1制备得到的具叶脉结构的GelMA别接种细胞（脐静脉血管内皮细胞，HUVECs）后，叠加，移植到裸鼠体内，3周后可见血管长入胶内，图12中黑色箭头指示为血管或血管网。其中，见图12A和12B为血管根部，图12C为血管末梢，图13为体内植入后第12周，可见裸鼠血管长入叶脉结构内，其走行与叶脉结构相似。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (1)

1. 一种仿生的预脉管化材料的制备方法，其特征在于，包括以下步骤：
步骤1. 制备具有叶脉结构的生物材料；
步骤2. 将步骤1中获得的生物材料消毒、清洗后，置于细胞培养基中静置过夜，然后接种细胞进行体外细胞培养；
将接种有细胞的互成镜像结构的生物材料叠加，得到具有中空结构的三维脉管化网络结构的预脉管化材料；
或，
将接种有细胞的具有叶脉结构的生物材料与无叶脉结构的生物材料叠加后，得到具有中空结构的三维脉管化网络结构的预脉管化材料；
或，
将消毒后的互成镜像结构的生物材料叠加，或者将消毒后的具有叶脉结构的生物材料与无叶脉结构的生物材料叠加后进行细胞灌注培养，得到具有中空结构的三维脉管化网络结构的预脉管化材料。
2. 根据权利要求1所述仿生的预脉管化材料的制备方法，其特征在于，步骤1.包括以下步骤：
步骤11. 将天然植物的叶脉结构置于培养皿或其他容器中，背面朝上，润湿，使其与容器底面贴合；
步骤12. 聚二甲基硅氧烷制膜：制备PDMS并将其覆盖步骤1中叶脉结构表面进行固化后，取下叶脉后得到具有叶脉“镜面结构”的PDMS薄膜；
步骤13. 聚二甲基硅氧烷倒模：制备PDMS并将其平铺于步骤2中的具有叶脉“镜面结构”的PDMS薄膜的表面进行固化后，取下具有叶脉“镜面结构”的PDMS薄膜，得到与叶脉相同结构的PDMS模板；
步骤14. 取GelMA溶液或明胶置于步骤3得到的PDMS模板的表面进行交联固化成型，然后取下PDMS模板，得具有叶脉结构的GelMA或明胶。
3. 根据权利要求1所述仿生的预脉管化材料的制备方法，其特征在于，步骤1包括以下步骤：
通过3D打印技术，以生物材料为“打印墨水”，以叶脉结构为模板，打印出具有叶脉结构的生物材料；
优选地，步骤1包括以下步骤：
以激光打印技术为基础，以固化的生物材料为基质，以叶脉结构为模板，打印出具有叶脉结构的生物材料。
4. 根据权利要求2所述仿生的预脉管化材料的制备方法，其特征在于，步骤11中所述天然植物的叶脉结构由以下任一方法制备得到：
方法一：将树叶置于8~12%氢氧化钠溶液中煮沸5~10分钟后，流水洗去残留氢氧化钠溶液，镊子轻刮或毛刷刷去叶肉，清洗后可获得叶脉；
方法二：将树叶放在水中煮沸5~10分钟后，流水洗去残留溶液，镊子轻刮或毛刷刷去叶肉，清洗后可获得叶脉；
方法三：将树叶置于3~5%氢氧化钠和2~5%无水碳酸钠的混合溶液中煮沸5~10分钟后，在清水中洗去腐蚀溶液，再用毛刷刷掉叶肉，清洗后可获得叶脉。
5. 根据权利要求2所述仿生的预脉管化材料的制备方法，其特征在于，所述制备PDMS的方法为：将硅酮和固化剂混合，两者混合比例8~12:1，除气泡后，于温度25-150℃下，固化反应10分钟-24小时。
6. 根据权利要求2所述仿生的预脉管化材料的制备方法，其特征在于，
步骤14中所述交联固化包括以下任一方法：
用磷酸盐缓冲液制备浓度5%~20% GelMA，与四甲基乙二胺混合，加入冰上预冷的过硫酸铵，迅速混匀后即平铺于步骤3得到的已于冰上或2~8℃预冷的PDMS模板的表面进行交联固化成型；
或，在浓度为0.01%~1%的Irgacure2959溶液中加入GelMA混合，取一定量混合液倒于由步骤S3中的PDMS模板表面，在UV灯下照射10~600秒，交联固化成型。
优选地，步骤2中的消毒方法包括以下任一方法：
具叶脉结构的GelMA或明胶置于在无菌环境中，室温下置于70%~75%酒精中浸泡30分钟，再使用无菌DPBS或PBS清洗1~5次，每次浸泡2~10分钟；
或，对具叶脉结构的GelMA或明胶进行紫外消毒或气体消毒；
或，对具叶脉结构的GelMA或明胶进行70%~75%酒精浸泡同时紫外消毒或气体消毒。
7. 根据权利要求1所述仿生的预脉管化材料的制备方法，其特征在于，步骤2中所述细胞、细胞培养基包括以下的一种或几种：
1）血管内皮细胞包括自体、异体或异种来源的脐静脉血管内皮细胞、微血管内皮细胞、内皮祖细胞；
血管内皮细胞完全培养基：内皮细胞基础培养基，5~10%FBS，1%双抗，血管生长因子；所述血管生长因子包括：促进内皮细胞分裂增殖和/或分化的因子或蛋白；
2）干细胞包括，自体、异体或异种来源的胚胎干细胞和成体干细胞，如脂肪干细胞、骨髓间充质干细胞、表皮干细胞、神经干细胞；
干细胞培养基：10%FBS，α-MEM、间充质干细胞培养基、高糖培养基和/或定向分化培养基，1%双抗；
3）成纤维细胞；
自体、异体或异种来源成纤维细胞培养基：DMEM，10%FBS，1%双抗；
4）自体、异体或异种来源诱导性多能干细胞，诱导性多能干细胞培养基：Essential 6™ Medium；
5) 自体、异体或异种来源的淋巴管内皮细胞；
淋巴管内皮细胞完全培养基：内皮细胞基础培养基，5-10%FBS，1%双抗，血管生长因子；所述血管生长因子包括促进内皮细胞分裂增殖和分化的因子或蛋白。
8. 根据权利要求1所述仿生的预脉管化材料的制备方法，其特征在于，所述血管生长因子包括：血管内皮细胞生长因子、成纤维细胞生长因子、血小板生长因子、转化生长因子β中的一种或几种。
9. 一种权利要求1~8所述制备方法制备得到的仿生的预脉管化材料。
10. 一种权利要求9所述仿生的预脉管化材料在制备创面和组织修复材料、支架材料、器官再造材料、组织替代材料、组织填充材料或包裹材料、原位诱导再生材料中的应用；
优选地，所述创面和组织修复材料、支架材料、器官再造材料、组织替代材料、组织填充材料或包裹材料、原位诱导再生材料包括：皮下填充材料、真皮组织萎缩或缺损填充材料、肌组织缺损填充或替代材料、乳房填充或替代材料、鼻填充或替代材料、臀部填充或替代材料、或假体包裹材料、人造血管化皮瓣、血管化人造组织、原位缝合替代材料、补充缺失或失活组织/器官的材料中的一种或几种。
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HE JIANKANG, MAO MAO, LIU YAXIONG, SHAO JINYOU, JIN ZHONGMIN, LI DICHEN: "Fabrication of Nature-Inspired Microfluidic Network for Perfusable Tissue Constructs", ADVANCED HEALTHCARE MATERIALS, WILEY - V C H VERLAG GMBH & CO. KGAA, DE, vol. 2, no. 8, 1 August 2013 (2013-08-01), DE, pages 1108 - 1113, XP055774864, ISSN: 2192-2640, DOI: 10.1002/adhm.201200404 *

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