WO2021217621A1 - 生物墨水、小口径管状结构支架及其制备方法和应用 - Google Patents

生物墨水、小口径管状结构支架及其制备方法和应用 Download PDF

Info

Publication number
WO2021217621A1
WO2021217621A1 PCT/CN2020/088427 CN2020088427W WO2021217621A1 WO 2021217621 A1 WO2021217621 A1 WO 2021217621A1 CN 2020088427 W CN2020088427 W CN 2020088427W WO 2021217621 A1 WO2021217621 A1 WO 2021217621A1
Authority
WO
WIPO (PCT)
Prior art keywords
tubular structure
small
ink
diameter
stent
Prior art date
Application number
PCT/CN2020/088427
Other languages
English (en)
French (fr)
Inventor
阮长顺
梁青飞
吴明明
Original Assignee
深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳先进技术研究院 filed Critical 深圳先进技术研究院
Priority to PCT/CN2020/088427 priority Critical patent/WO2021217621A1/zh
Publication of WO2021217621A1 publication Critical patent/WO2021217621A1/zh

Links

Images

Classifications

    • 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
    • A61L27/02—Inorganic 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
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • 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
    • B33Y70/00—Materials specially adapted for additive manufacturing

Definitions

  • This application relates to the technical field of biomedical materials, in particular to bio-inks, small-diameter tubular structure stents, and preparation methods and applications thereof.
  • 3D printing technology is widely used in construction, aerospace, automotive industry and other fields. With the development of biotechnology, 3D printing plays an increasingly important role in the field of biomedicine.
  • the use of 3D coaxial printing technology to construct a three-dimensional tubular structure stent can be used to simulate the human urinary tube, intestinal tube, esophagus, trachea, bile duct, blood vessel and other tubular tissues, which is of great significance in the medical field.
  • Bio-ink is the main raw material for 3D printing.
  • the technology for preparing large-caliber tubular structure stents is relatively mature, which can meet clinical needs to a certain extent.
  • the small-caliber tubular structure stent often has the disadvantage of poor mechanical performance, especially under long-term liquid fluid shear, its structural stability tends to be greatly reduced, which greatly limits its use in regenerative medicine and drug toxicology research.
  • the traditional agarose template sacrifice method, electrospinning method, stereo lithography method, self-assembly method and microfluidic technology, etc. also have the disadvantages of complicated operation, low precision, and difficulty in rapid customization, and the fabricated tube
  • the structural scaffold has poor mechanical properties, poor structural stability and low biocompatibility activity.
  • the embodiments of the present application provide a biological ink, a small-diameter tubular structure stent, and a preparation method and application thereof.
  • the biological ink has a simple formula, and the composite material network formed by curing the biological ink has a high degree of cross-linking and a structure Stable, strong mechanical properties, and high biocompatibility activity.
  • the present application provides a biological ink for 3D printing, including N-acryloylglycinamide (NAGA), high molecular polymer and nano clay (Clay), wherein the high molecular polymer includes One or more of modified gelatin, double bond modified alginate, double bond modified collagen, and double bond modified hyaluronic acid; the N-acryloyl glycinamide and the polymer The mass ratio is (0.1-10):1.
  • NAGA N-acryloylglycinamide
  • Clay nano clay
  • the bio-ink described in this application is in the form of a hydrogel.
  • the bio-ink is composed of N-acryloylglycinamide, high molecular polymer, nanoclay and the balance of water.
  • the side chain of the N-acryloylglycinamide has two amide groups.
  • the N-acryloylglycinamide can be prepared from glycinamide hydrochloride and acryloyl chloride as raw materials.
  • the modified gelatin is methacrylic anhydride modified gelatin (GelMA).
  • the gelatin is derived from natural gelatin, for example, animal gelatin.
  • the gelatin is derived from porcine skin gelatin.
  • the double bond grafting rate of the modified gelatin is greater than 70%.
  • the double bond grafting rate of the modified gelatin is 70-85%.
  • the double bond grafting rate of the modified gelatin is 81-85%.
  • the double bond grafting rate of the modified gelatin is 70%, 75%, 78%, 81%, 82%, 83%, 84% or 85%.
  • the molecular weight of the modified gelatin is greater than 12-14 kDa. In one embodiment, the molecular weight of the modified gelatin is 15-60 kDa.
  • the modified gelatin with the double bond grafting rate range and molecular weight described in the present application is beneficial for the formed bio-ink to have a higher degree of network crosslinking after curing, and the structure is more stable.
  • the mass ratio of the N-acryloylglycinamide and modified gelatin is (1-10):1.
  • the mass ratio of the N-acryloylglycinamide and modified gelatin is 1:9, 3:7, 1:1, 5:5, 7:3, 9:1 or 10:1.
  • the nano-clay is in the shape of nano-sheets, and the lateral size of the nano-clay is 20-40 nm; and the thickness is 0.5-5 nm.
  • the lateral dimension of the nano-clay is 25-45 nm; the thickness is 0.5-3 nm.
  • the mass percentage content of the N-acryloylglycinamide in the bio-ink is 10%-30%. In one embodiment, the mass percentage of the N-acryloylglycinamide in the bio-ink is 12%-26%. In another embodiment, the mass percentage of the N-acryloylglycinamide in the bio-ink is 20%-26%. For example, the mass percentage of the N-acryloylglycinamide in the bio-ink is 10%, 12%, 15%, 18%, 20%, 25%, 28%, 29% or 30%.
  • the mass percentage of the high molecular polymer in the bio-ink is 1%-16%. In one embodiment, the mass percentage of the high molecular polymer in the bio-ink is 2%-15%. In another embodiment, the mass percentage of the high molecular polymer in the bio-ink is 5%-15%. In the third embodiment, the mass percentage of the high molecular polymer in the bio-ink is 10%-15%. For example, the mass percentage of the high molecular polymer in the biological ink is 1%, 2%, 5%, 8%, 10%, 12%, 13%, 15% or 16%.
  • the high molecular polymer when the bio-ink is cured and cross-linked, the high molecular polymer can participate in the cross-linking; among them, modified gelatin, double bond modified alginate, double bond modified collagen or double bond modified hyaluronic acid Cross-linking occurs through double bonds in the molecule.
  • the mass percentage of the nano-clay in the bio-ink is 3-10%.
  • the mass percentage of the nano-clay in the bio-ink is 4-10%.
  • the mass percentage of the nano-clay in the bio-ink is 5-8%.
  • the mass percentage of the nano clay is 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.
  • the biological ink further includes a photoinitiator, and the mass percentage of the photoinitiator in the biological ink is 0.1%-0.5%.
  • the bio-ink is composed of N-acryloylglycinamide, high molecular polymer, nano-clay, photoinitiator and balance water.
  • the quality percentage of the photoinitiator is 0.2-0.4%.
  • the mass percentage of the photoinitiator is 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.4% or 0.5%.
  • the photoinitiator can accelerate the cross-linking and curing of the biological ink under light irradiation, and the photoinitiator in the content range can promote the cross-linking and curing of the biological ink at an appropriate speed under light irradiation to form a mechanical
  • the photoinitiator is photoinitiator 1173 (2-Hydroxy-2-methylpropiophenone).
  • the biological ink described in the first aspect of the present application can be used for 3D printing.
  • the biological ink has a simple formula and simple preparation, and can be suitable for industrial production.
  • the composite material network formed by curing the bio-ink has high cross-linking degree, stable structure, strong mechanical properties, and high biocompatibility activity.
  • the present application provides a small-caliber tubular structure stent, which is prepared by 3D printing from the bio-ink described in the first aspect of the present application. Based on different 3D printing processes, the specific shape of the small-diameter tubular structure stent described in this application can be adjusted.
  • the small-caliber tubular structure stent includes at least one hollow tubular structure, and the tube wall of the hollow tubular structure includes a single layer or multiple layers of composite material, and the composite material layer is formed by curing the biological ink.
  • the small-diameter tubular structure stent is a hollow tubular structure.
  • the tube wall of the small-diameter tubular structure stent may include, but is not limited to, a single layer, a double layer, a three layer, or a composite material layer with more than three layers.
  • Each layer of the composite material may be the same or different.
  • the difference in the composite material layer means that it is formed by cross-linking and curing of bio-inks with different content ratios.
  • the tube wall of the hollow tubular structure includes an inner layer, an intermediate layer, and an outer composite material layer that are sequentially stacked from the inside to the outside.
  • the inner diameter of the small-diameter tubular structure stent is 0.1-2.8 mm, and the outer diameter of the small-diameter tubular structure stent is 0.5-6.0 mm.
  • the inner diameter of the small-diameter tubular structure stent is 0.1-2.0 mm
  • the outer diameter of the small-diameter tubular structure stent is 0.5-3.0 mm.
  • the small-caliber tubular structure stent described in the present application has a small inner diameter and an outer diameter, and has good mechanical properties.
  • the tensile breaking strength of the small-diameter tubular structure stent is 10-30 MPa; the elongation rate of the small-diameter tubular structure stent is 40-500%.
  • the tensile breaking strength of the small-diameter tubular structure stent is 15-25 MPa.
  • the tensile breaking strength of the small-caliber tubular structure stent is 10 MPa, 15 MPa, 18 MPa, 20 MPa, 25 MPa or 30 MPa.
  • the stretch rate of the small-diameter tubular structure stent is 100-500%.
  • the stretch rate of the small-caliber tubular structure stent is 200-500%.
  • the stretch rate of the small-caliber tubular structure stent is 40%, 80%, 100%, 200%, 250%, 300% or 400% or 500%.
  • the small-caliber tubular structure stent described in the present application has outstanding mechanical properties, good elongation rate and tensile breaking strength, and strong tensile resistance.
  • the suture retention strength of the small-caliber tubular structure stent is 80-300 grams force (GF).
  • the suture retention strength of the small-caliber tubular structure stent is 100-300 grams force.
  • the suture thread of the small-caliber tubular structure stent described in the present application has high retention strength and strong mechanical properties, and is suitable for suture thread.
  • the fatigue resistance of the small-diameter tubular structure stent is outstanding.
  • the small-caliber tubular structure stent can withstand more than 500 cyclic stress tests.
  • the small-caliber tubular structure stent described in the present application also has good blast resistance.
  • the small-diameter tubular structure stent maintains a small diameter (inner and outer diameter) while still maintaining good mechanical strength, high suture retention strength, outstanding tensile properties, and excellent Outstanding anti-fatigue performance. Since the small-diameter tubular structure stent also has good biocompatibility activity, the small-diameter tubular structure stent can be widely used in tissue engineering fields including artificial blood vessels.
  • this application also provides a method for preparing a small-caliber tubular structure stent, which includes the following steps:
  • the high molecular polymer includes modified gelatin, double bond modified alginate, and double bond modified collagen And one or more of hyaluronic acid modified with double bonds, the mass ratio of the N-acryloylglycinamide to the high molecular polymer is (0.1-10):1;
  • bio-ink is filled into the printing material cylinder, and the 3D coaxial printing process is used to print in a preset size, and then irradiated by light. After cross-linking and curing, a small-diameter tubular structure stent is obtained.
  • the 3D coaxial printing process refers to using one or more materials from different pipes to flow out of a tubular nozzle with a coaxial structure at the same time to solidify, so as to form a tubular structure stent.
  • 3D printing is performed by a 3D coaxial printer to construct the small-diameter tubular structure stent.
  • the mass percentage content of the N-acryloylglycinamide in the bio-ink is 10%-30%. In one embodiment, the mass percentage of the N-acryloylglycinamide in the bio-ink is 12%-26%. In another embodiment, the mass percentage of the N-acryloylglycinamide in the bio-ink is 20%-26%. For example, the mass percentage of the N-acryloylglycinamide in the bio-ink is 10%, 12%, 15%, 18%, 20%, 25%, 28%, 29% or 30%.
  • the mass percentage of the high molecular polymer in the bio-ink is 1%-16%. In one embodiment, the mass percentage of the high molecular polymer in the bio-ink is 2%-15%. In another embodiment, the mass percentage of the high molecular polymer in the bio-ink is 5%-15%. In the third embodiment, the mass percentage of the high molecular polymer in the bio-ink is 10%-15%. For example, the mass percentage of the high molecular polymer in the biological ink is 1%, 2%, 5%, 8%, 10%, 12%, 13%, 15% or 16%.
  • the modified gelatin is methacrylic anhydride modified gelatin.
  • the double bond grafting rate of the modified gelatin is greater than 70%.
  • the double bond grafting rate of the modified gelatin is 70-85%.
  • the double bond grafting rate of the modified gelatin is 81-85%.
  • This application uses modified gelatin with a high double bond grafting rate, which can greatly improve the mechanical properties of the prepared small-diameter tubular structure scaffold.
  • the mass percentage of the nano-clay in the bio-ink is 3-10%.
  • the mass percentage of the nano-clay in the bio-ink is 4-10%.
  • the mass percentage of the nano-clay in the bio-ink is 5-8%.
  • the mass percentage of the nano clay is 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.
  • the surface of which has positive and negative charges is the surface of which has positive and negative charges.
  • the polymer materials of the stent are cross-linked through physical and chemical methods, which greatly improves the mechanical properties of the final small-diameter tubular structure stent, the structure is more stable, and the service life of the small-diameter tubular structure stent is extended.
  • the diameter (outer diameter and inner diameter) of the small-diameter tubular structure stent can be flexibly adjusted during the preparation process.
  • the desired size can be obtained by adjusting the printing extrusion pressure and the printing line moving speed.
  • the solid content or viscosity of the bio-ink is different; when the viscosity of the bio-ink is higher, the pressure required for printing is higher; when the viscosity is lower, the pressure for printing is also reduced.
  • the moving speed of the printed line is inversely proportional to the diameter (outer diameter and inner diameter). For example, the higher the moving speed, the thinner the line; and vice versa.
  • the inner diameter of the semi-finished small-diameter tubular structure stent is 0.1-2.8 mm, and the outer diameter of the small-diameter tubular structure stent is 0.5-6 mm.
  • an extrusion pressure of 80-200 kPa is used to extrude the small-diameter tubular structure stent from the 3D printing device.
  • an extrusion pressure of 80-150 kPa is used to extrude the small-caliber tubular structure stent from the 3D printing device.
  • ultraviolet light is used for irradiation
  • the center wavelength of the ultraviolet light is 360-370 nm
  • the crosslinking time is 0.25-60 min.
  • a UV crosslinker device is used for UV crosslinking.
  • the crosslinking time can also be 1-20 min. Based on different intensities of UV light, the cross-linking time is adjusted.
  • the obtained small-diameter tubular structure stent is soaked in a buffer solution, and after the swelling balance of the small-diameter tubular structure stent is balanced, the performance test is performed on the small-diameter tubular structure stent after the swelling balance.
  • the buffer can be, but is not limited to, a PBS buffer.
  • the small-caliber tubular structure stent after swelling and balance can make the performance test data more reliable.
  • the preparation method described in the third aspect of the present application can prepare a high-strength small-diameter tubular structure stent with both dimensional controllability and mechanical adjustability in one step; the preparation method is simple in production method, low in cost, and suitable for industrial production .
  • this application also provides a small-diameter tubular structure stent as described in the second aspect of the application or the small-diameter tubular structure prepared by the preparation method in the third aspect of the application in artificial tissues, drug screening and pathology. Application in the model.
  • the size of the small-caliber tubular structure described in this application is controllable, and the small-caliber tubular structure has outstanding mechanical properties and good biocompatibility, and can be used to simulate human urinary tubes, intestinal tubes, esophagus, trachea, bile ducts, blood vessels, etc. Tissues have broad application prospects in artificial tissues, drug screening and pathological model research.
  • the small-diameter tubular structure can be smaller in size while still maintaining outstanding mechanical properties
  • the small-diameter tubular structure especially has outstanding mechanical properties and is widely used in the field of biomedicine or tissue engineering.
  • the small-caliber tubular structure can be used to solve the problems of lack of donor sources for human tissue and organ transplantation and immune rejection.
  • Fig. 1 is a hydrogen spectrum chart of NAGA monomer provided by an embodiment of the application.
  • Figure 2 is a hydrogen spectrum of gelatin and GelMA provided in an embodiment of the application
  • Figure 3 is an infrared spectrogram of CNG bio-ink provided by an embodiment of the application.
  • FIG. 4 is a schematic diagram of 3D printing preparation of a small-diameter tubular structure stent provided by an embodiment of the application;
  • Fig. 5 is a diagram of actual samples of a small-diameter tubular structure stent with different diameters provided by an embodiment of the application;
  • Fig. 6 is a scanning electron microscope diagram of a small-diameter tubular structure stent provided by an embodiment of the application;
  • Fig. 7 is a diagram showing the tensile performance of a small-diameter tubular structure stent provided by an embodiment of the application.
  • Fig. 8 is a test diagram of burst pressure and fracture suture of a small-caliber tubular structure stent provided by an embodiment of the application;
  • FIG. 9 is a test diagram of fatigue resistance performance of a small-diameter tubular structure stent provided by an embodiment of the application.
  • An embodiment of the present application provides a method for preparing a small-diameter tubular structure stent.
  • the pork skin gelatin was mixed into PBS buffer at a ratio of 10% (w/v), and stirred under heating in a water bath at 50° C. until the ingredients were completely dissolved. Subsequently, 8 mL (v/v) of methacrylic anhydride was added dropwise to the gelatin solution, and reacted at 50° C. for 3 hours to form a GelMA solution.
  • the solution was diluted and dialyzed with distilled water in a dialysis bag with a molecular weight of 12-14kDa at 40°C for one week to remove small molecular weight products and reactants in the solution; the modified gelatin solution obtained after dialysis was lyophilized for 4 days to produce The white porous foamy product was then stored at -80°C for later use.
  • Figure 2 shows the proton nuclear magnetic resonance spectra of gelatin and modified gelatin (GelMA).
  • GelMA modified gelatin
  • the proton peak of the bond indicates that the double bond has been successfully connected to the gelatin molecular chain, that is, GelMA has been successfully synthesized.
  • the GelMA double bond graft rate was measured by 1 H NMR (NMR, 500 MHz, Varian INOVA). Gelatin and modified gelatin were dissolved in D 2 O at a concentration of 10 mg/mL. The results showed that the double bond grafting rate in modified gelatin was about 81%.
  • the mass ratio of NAGA/GelMA can be 1:9, 3:7, 5:5, 7:3, 9:1.
  • the mass content of the NAGA, GelMA, Clay and photoinitiator in the CNG bio-ink can be 10%-30%, 1%-16%, 3%-10% and 0.1%-0.5%, respectively.
  • the characteristic peak of GelMA shows the characteristic peak of the gelatin main chain (B).
  • the characteristic band at 1540 cm -1 is attributed to the stretching vibration of the NH band (amide II), and the characteristic peak at 1250 cm -1 is attributed to the CN stretching vibration of the amino acid side chain (amide III).
  • the spectrum of Clay/NAGA/GelMA hydrogel in addition to the characteristic peaks of NAGA and GelMA, there are also characteristic peaks of nanoclay (Clay). Among them, the Si-O stretching vibration and bending vibration peaks appear at 1006 cm -1 and 660 cm -1 . This result indicates that the NAGA/GelMA/Clay hybrid hydrogel was successfully crosslinked.
  • the CNG bio-ink prepared above is filled in the material cylinder, and the semi-finished product of the small-diameter tubular structure stent with controllable size is extruded through a 3D printing device (such as a 3D coaxial printer) under a pressure of 80-200kpa Finally, put it into an ultraviolet cross-linking instrument, and cross-link it for a specific time (0.25-60 min) under a specific ultraviolet intensity (the center wavelength is 365 nm). After fully cross-linking, a small-diameter tubular structure stent is obtained.
  • nano-clay particles can achieve physical interpenetration and thickening effects, both through physical interpenetration and chemical cross-linking to improve the mechanical stability and biological activity of the small-diameter tubular structure scaffold.
  • Example 1 A method for preparing a small-caliber tubular structure stent, including the following steps:
  • CNG-01 Clay/NAGA/GelMA mixed hydrogel Ink
  • the CNG-01 mixed hydrogel bio-ink is loaded into the printing material cylinder, and a hollow tubular structure with a controllable size is extruded through a 3D coaxial printer under a pressure of 80-100kPa (outer diameter OD is 0.5mm-3mm, The inner diameter ID is 0.1-2.8mm), and finally it is put into an ultraviolet cross-linking instrument, and cross-linked for 40 minutes at a specific ultraviolet intensity with a central wavelength of 365 nm to obtain a small-diameter tubular structure stent sample.
  • Embodiment 2 A method for preparing a small-caliber tubular structure stent, including the following steps:
  • CNG-02 Clay/NAGA/GelMA mixed hydrogel organism Ink
  • the CNG-02 mixed hydrogel bio-ink is loaded into the printing material cylinder, and a hollow tubular structure with a controllable size is extruded through a 3D coaxial printer under a pressure of 100-120kPa (outer diameter OD is 0.5mm-3mm, The inner diameter ID is 0.1-2.8mm), and finally it is put into an ultraviolet cross-linking instrument, and cross-linked for 40 minutes at a specific ultraviolet intensity with a central wavelength of 365 nm to obtain a small-diameter tubular structure stent sample.
  • Embodiment 3 A method for preparing a small-caliber tubular structure stent, including the following steps:
  • the CNG-03 mixed hydrogel bio-ink is loaded into the printing material cylinder, and a hollow tubular structure with a controllable size is extruded through a 3D coaxial printer under a pressure of 100-130kPa (outer diameter OD is 0.5mm-3mm, The inner diameter ID is 0.1-2.8mm), and finally it is put into an ultraviolet cross-linking instrument, and cross-linked for 40 minutes at a specific ultraviolet intensity with a central wavelength of 365 nm to obtain a small-diameter tubular structure stent sample.
  • Embodiment 4 A method for preparing a small-caliber tubular structure stent, including the following steps:
  • Hyaluronan double bond-modified hyaluronic acid (Hyaluronan, abbreviated HA) in 1 mL of deionized water to fully dissolve;
  • Clay/NAGA/double bond modified HA hybrid hydrogel bio-ink referred to as CNH-01 (where the mass of Clay is 100 mg, and the mass ratio of NAGA to double bond modified HA is 5:5).
  • Embodiment 5 A method for preparing a small-diameter tubular structure stent, including the following steps:
  • Embodiment 6 A method for preparing a small-caliber tubular structure stent, including the following steps:
  • the preparation method described in this application can be used to prepare different diameters (the outer diameter OD is 0.5mm-3mm, and the inner diameter ID is 0.1-2.8mm).
  • the small-caliber tubular structure stent is produced with a stable structure.
  • the small-diameter tubular structure stent CNG-01 prepared in Example 1 has a tensile breaking strength ( ⁇ 22MPa) and an elongation rate ( ⁇ 500%) when the outer diameter/inner diameter is about 3mm/2.4mm; 2 The tensile breaking strength ( ⁇ 10MPa) and the elongation rate ( ⁇ 60%) of the prepared small-diameter tubular structure stent CNG-02; the small-diameter tubular structure stent CNG-03 prepared in Example 3 ( ⁇ 8MPa), elongation ( ⁇ 43%).
  • the small-diameter tubular structure stent prepared in the examples of the present application has an elongation rate of 43-500% and excellent mechanical properties.
  • the Young's modulus of each embodiment was tested, and the results also showed that the Young's modulus of Examples 1-3 could reach 35MPa, 24MP and 21MPa, respectively.
  • Burst pressure test is carried out in accordance with the national standard AS ISO7198-2003.
  • the anti-blasting performance of the tubular structure bracket was tested through the self-made anti-blasting pressure device platform.
  • the hollow tube is fixed to the device with 6-0 silk thread, and the pressure is increased at a rate of 50mmHg s -1 until the microtube ruptures .
  • the fracture suture test was carried out in accordance with ASISO 7198-2003.
  • the total length of the small-caliber tubular structure stent sample is about 3 cm. Fix one end of the tubular structure bracket to the bench clamp of the uniaxial tensile testing machine. At a distance of 3mm from the edge, use a 6-0 polypropylene suture needle through the wall of the tube, and pull and suture at a constant rate of 50mm/min. Thread until the specimen is completely torn; the maximum force is recorded as the suture retention strength. Take the small-diameter tubular structure stents of each outer diameter/inner diameter prepared in Example 1 and Example 2 for testing. Before the test, the samples of each group were soaked in PBS at 37° C. overnight. The results are shown in FIG. 8.
  • groups 4-6 are the small-diameter tubular structure stent samples with NAGA/GelMA mass ratio 7:3 in Example 2.
  • the outer diameter/inner diameter are 2.2mm/0.3mm, 2.8mm/0.1, respectively. mm, 3mm/0.5mm.
  • Groups 7-10 are the small-diameter tubular structure stent samples with a mass ratio of NAGA/GelMA of 9:1 in Example 1.
  • the outer diameter/inner diameter are 2.2mm/0.3mm, 2.2mm/0.3mm, 2.8mm/ 0.1mm, 3mm/0.3mm.
  • B and Groups 2-4 are the small-diameter tubular structure stent samples with NAGA/GelMA mass ratio 7:3 in Example 2.
  • the outer diameter/inner diameter are 2.2mm/0.3mm, 2.8mm/0.1, respectively. mm, 3mm/0.5mm; Groups 5-8 are the small-diameter tubular structure stent samples with a mass ratio of NAGA/GelMA of 9:1 in Example 1.
  • the outer diameter/inner diameter are 2.2mm/0.3mm, 2.2mm, respectively /0.3mm, 2.8mm/0.1mm, 3mm/0.3mm.
  • the burst pressure of the small-caliber tubular structure stent sample of Example 1 of the present application can reach about 1900 mmHg, and the suture retention strength can be 280 gf.
  • the burst pressure of the small-caliber tubular structure stent sample of Example 2 can reach about 2500 mmHg, and the suture retention strength can be 85 gf.
  • the small-caliber tubular structure stent sample prepared in the examples of this application has excellent burst resistance, high suture retention strength, and its burst pressure is comparable to or even better than the existing aortic tube, esophageal tube and tracheal parameters; its suture line maintains
  • the strength can also reach 280gf, showing excellent structural stability and mechanical properties.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Dermatology (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Manufacturing & Machinery (AREA)
  • Materials Engineering (AREA)
  • Inorganic Chemistry (AREA)
  • Materials For Medical Uses (AREA)

Abstract

一种用于3D打印的生物墨水，包括N-丙烯酰基甘氨酰胺、高分子聚合物和纳米黏土。高分子聚合物包括改性明胶、双键修饰的海藻酸盐、双键修饰的胶原和双键修饰的透明质酸中的一种或多种，N-丙烯酰基甘氨酰胺和高分子聚合物的质量比为（0.1-10）：1。生物墨水固化形成的复合材料网络交联度高、结构稳定、机械性能强，具有高生物相容性。一种由该生物墨水经3D打印形成的小口径管状结构支架及其制备方法和应用。

Description

生物墨水、小口径管状结构支架及其制备方法和应用 技术领域
本申请涉及生物医学材料技术领域，特别是涉及生物墨水、小口径管状结构支架及其制备方法和应用。
背景技术
3D打印技术在建筑、航天、汽车工业等领域得到广泛应用。随着生物科技发展，3D打印在生物医学领域中发挥的作用也愈加重要。例如，利用3D同轴打印技术构建三维管状结构支架可用以模拟人体尿管、肠管、食管、气管、胆管、血管等管状组织，在医学领域具有重要意义。生物墨水是3D打印的主要原料。
目前，关于制备大口径管状结构支架(Φ>6mm)的技术相对比较成熟，在一定程度上能够满足临床需求。然而对于小口径管状结构支架往往存在机械性能差的缺点，尤其是在长期处于液体流体剪切力下，其结构稳定性往往会大幅降低，极大地限制了其在在再生医学、药物毒理学研究和基础研究的应用和发展。而传统琼脂糖模板牺牲法、静电纺丝法、立体光刻法、自主装法以及微流控技术等，也存在操作复杂、精度低及快速个性化定制难度大等缺点，且制得的管状结构支架的机械性能差、结构稳定性差及生物相容活性低等。
发明内容
有鉴于此，本申请实施例提供了一种生物墨水、小口径管状结构支架及其制备方法和应用，该生物墨水配方简单，由所述生物墨水固化形成的复合材料网络交联度高，结构稳定，机械性能强，以及具有高生物相容活性。
第一方面，本申请提供了一种生物墨水，用于3D打印，包括N-丙烯酰基甘氨酰胺(NAGA)、高分子聚合物和纳米黏土(Clay)，其中，所述高分子聚合物 包括改性明胶、双键修饰的海藻酸盐、双键修饰的胶原和双键修饰的透明质酸中的一种或多种；所述N-丙烯酰基甘氨酰胺和所述高分子聚合物的质量比为(0.1-10):1。
本申请所述生物墨水呈水凝胶状。可选地，所述生物墨水由N-丙烯酰基甘氨酰胺、高分子聚合物、纳米黏土和余量水组成。
本申请实施方式中，所述N-丙烯酰基甘氨酰胺的侧链带有两个酰胺基团。其中，所述N-丙烯酰基甘氨酰胺可以甘氨酰胺盐酸盐和丙烯酰氯为原材料制备获得。
可选地，所述改性明胶为甲基丙烯酸酐改性明胶(GelMA)。其中，所述明胶来源于天然明胶，例如，动物明胶。一实施方式中，所述明胶来源于猪皮明胶。
可选地，所述改性明胶的双键接枝率大于70％。可选地，所述改性明胶的双键接枝率为70-85％。可选地，所述改性明胶的双键接枝率为81-85％。例如，所述改性明胶的双键接枝率为70％，75％，78％，81％，82％，83％，84％或85％。
可选地，所述改性明胶的分子量大于12-14kDa。一实施方式中，所述改性明胶的分子量为15-60kDa。本申请所述双键接枝率范围和分子量的改性明胶有利于形成的生物墨水固化后具备更高的网络交联度，结构更稳定。
可选地，所述N-丙烯酰基甘氨酰胺和改性明胶的质量比为(1-10):1。例如，所述N-丙烯酰基甘氨酰胺和改性明胶的质量比为1:9，3:7，1:1，5:5，7:3，9:1或10:1。
本申请实施方式中，所述纳米黏土为纳米片状，所述纳米黏土的横向尺寸为20-40nm；厚度为0.5-5nm。可选地，所述纳米黏土的横向尺寸为25-45nm；厚度为0.5-3nm。
可选地，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量为10％-30％。一实施方式中，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量为12％-26％。另一实施方式中，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量为20％-26％。例如，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量10％、12％、15％、18％、20％、25％、28％、29％或30％。
可选地，所述高分子聚合物在所述生物墨水中的质量百分含量为1％-16％。一实施方式中，所述高分子聚合物在所述生物墨水中的质量百分含量为2％-15％。另一实施方式中，所述高分子聚合物在所述生物墨水中的质量百分含量为5％-15％。第三实施方式中，所述高分子聚合物在所述生物墨水中的质量百分含量为10％-15％。例如，所述高分子聚合物在所述生物墨水中的质量百分含量1％、2％、5％、8％、10％、12％、13％、15％或16％。本申请中，当所述生物墨水固化交联时，高分子聚合物能参与交联；其中，改性明胶、双键修饰的海藻酸盐、双键修饰的胶原或双键修饰的透明质酸通过分子中的双键进行交联。
可选地，所述纳米黏土在所述生物墨水中的质量百分含量为3-10％。可选地，所述纳米黏土在所述生物墨水中的质量百分含量为4-10％。或者，所述纳米黏土在所述生物墨水中的质量百分含量为5-8％。本申请一具体实施方式中，所述纳米黏土的质量百分含量为3％、4％、5％、6％、7％、8％、9％或10％。本本申请所述生物墨水中的纳米黏土可以提升所述生物墨水固化后的网路交联度和机械性能。
可选地，所述生物墨水还包括光引发剂，所述光引发剂在所述生物墨水中的质量百分含量为0.1％-0.5％。例如，所述生物墨水由N-丙烯酰基甘氨酰胺、高分子聚合物、纳米黏土、光引发剂和余量水组成。可选地，所述光引发剂的质 量百分含量为0.2-0.4％。本申请一具体实施方式中，所述光引发剂的质量百分含量为0.1％、0.15％、0.2％、0.25％、0.3％、0.4％或0.5％。所述光引发剂可以使所述生物墨水在光照射下加速交联固化，其中，所述含量范围的光引发剂可以促使所述生物墨水在光照射下以适宜速度交联固化，以形成机械性能良好的复合材料，避免交联固化速度过快而导致复合材料机械性能减弱，抗疲劳性能差。例如，所述光引发剂为光引发剂1173(2-Hydroxy-2-methylpropiophenone)。
本申请第一方面所述的生物墨水能用于3D打印，该生物墨水该生物墨水配方简单，制备简单，可以适用于工业化生产。由所述生物墨水固化形成的复合材料网络交联度高，结构稳定，机械性能强，以及具有高生物相容活性。
第二方面，本申请提供了一种小口径管状结构支架，所述小口径管状结构支架由本申请第一方面所述生物墨水经3D打印制备得到。基于不同3D打印工艺，本申请所述小口径管状结构支架的具体形状可以进行调节。
可选地，所述小口径管状结构支架包括至少一根空心管状结构，所述空心管状结构的管壁包括单层或多层复合材料层，所述复合材料层由所述生物墨水固化形成。一实施方式中，所述小口径管状结构支架为一空心管状结构。
本申请实施方式中，所述小口径管状结构支架的管壁可以但不限于包括单层、双层、三层或三层以上的复合材料层。每层所述复合材料层可以相同也可以不同。所述复合材料层不同是指由不同含量配比的生物墨水交联固化形成。一实施方式中，所述空心管状结构的管壁包括从内向外依次层叠的内层、中间层和外层复合材料层。
本申请实施方式中，所述小口径管状结构支架的内径为0.1-2.8mm，所述小口径管状结构支架的外径为0.5-6.0mm。
可选地，所述小口径管状结构支架的内径为0.1-2.0mm，所述小口径管状结 构支架的外径为0.5-3.0mm。本申请所述小口径管状结构支架的内径和外径尺寸小，且具备良好的机械性能。
本申请实施方式中，所述小口径管状结构支架的拉伸断裂强度为10-30MPa；所述小口径管状结构支架的拉伸率为40-500％。可选地，所述小口径管状结构支架的拉伸断裂强度为15-25MPa。例如，所述小口径管状结构支架的拉伸断裂强度为10MPa、15MPa、18MPa、20MPa、25MPa或30MPa。可选地，所述小口径管状结构支架的拉伸率100-500％。或者，所述小口径管状结构支架的拉伸率为200-500％。例如，所述小口径管状结构支架的拉伸率为40％、80％、100％、200％、250％、300％或400％或500％。本申请所述小口径管状结构支架的机械性能突出，具有良好的拉伸率和拉伸断裂强度，抗拉伸性能强。
本申请实施方式中，所述小口径管状结构支架的缝合线保持强度为80-300克力(GF)。可选地，所述小口径管状结构支架的缝合线保持强度为100-300克力。本申请所述小口径管状结构支架的缝合线保持强度高，机械性能强，可适用于缝合线。
本申请实施方式中，所述小口径管状结构支架的抗疲劳性能突出。所述小口径管状结构支架可承受500次以上的循环应力测试。本申请所述小口径管状结构支架同样具有良好的抗爆破性能。
本申请第二方面所述小口径管状结构支架在维持较小直径(内、外径)尺寸条件下，仍保持良好的机械强度高，缝合线保持强度高，拉伸性能突出，且具有出色的抗疲劳性能突出。由于所述小口径管状结构支架还具有良好的生物相容活性，因此所述小口径管状结构支架可广泛用于包括人工血管等组织工程领域。
第三方面，本申请还提供了一种小口径管状结构支架的制备方法，包括以下 步骤：
配制高分子聚合物水溶液，加入N-丙烯酰基甘氨酰胺和纳米黏土，混合均匀后得到混合料，所述高分子聚合物包括改性明胶、双键修饰的海藻酸盐、双键修饰的胶原和双键修饰的透明质酸中的一种或多种，所述N-丙烯酰基甘氨酰胺和所述高分子聚合物的质量比为(0.1-10):1；
向所述混合料中加入光引发剂，避光搅拌均匀后，得到生物墨水，将所述生物墨水填装至打印物料筒中，采用3D同轴打印工艺按预设尺寸进行打印，然后经光照射交联固化后，得到小口径管状结构支架。
其中，所述3D同轴打印工艺是指利用一种或多种材料从不同管道同时流出一个具有同轴结构的管状喷头发生固化，以形成管状结构支架。本申请中，一实施方式中，通过3D同轴打印机进行3D打印，构建所述小口径管状结构支架。
可选地，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量为10％-30％。一实施方式中，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量为12％-26％。另一实施方式中，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量为20％-26％。例如，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量10％、12％、15％、18％、20％、25％、28％、29％或30％。
可选地，所述高分子聚合物在所述生物墨水中的质量百分含量为1％-16％。一实施方式中，所述高分子聚合物在所述生物墨水中的质量百分含量为2％-15％。另一实施方式中，所述高分子聚合物在所述生物墨水中的质量百分含量为5％-15％。第三实施方式中，所述高分子聚合物在所述生物墨水中的质量百分含量为10％-15％。例如，所述高分子聚合物在所述生物墨水中的质量百分含量1％、2％、5％、8％、10％、12％、13％、15％或16％。
本申请实施方式中，所述改性明胶为甲基丙烯酸酐改性明胶。可选地，所述改性明胶的双键接枝率大于70％。可选地，所述改性明胶的双键接枝率为70-85％。可选地，所述改性明胶的双键接枝率为81-85％。本申请使用双键接枝率高的改性明胶，可以大大提升制得的小口径管状结构支架机械性能。
可选地，所述纳米黏土在所述生物墨水中的质量百分含量为3-10％。可选地，所述纳米黏土在所述生物墨水中的质量百分含量为4-10％。或者，所述纳米黏土在所述生物墨水中的质量百分含量为5-8％。本申请一具体实施方式中，所述纳米黏土的质量百分含量为3％、4％、5％、6％、7％、8％、9％或10％。本申请所述纳米黏土，与N-丙烯酰基甘氨酰胺、高分子聚合物的交联过程中，其表面分布有正、负电荷，与所述N-丙烯酰基甘氨酰胺、高分子聚合物的聚合材料通过物理和化学两种方式参与交联，大大提升最后小口径管状结构支架的机械性能，结构更稳定，延伸小口径管状结构支架的使用寿命。
本申请实施方式中，所述小口径管状结构支架的直径(外径和内径)可以在制备过程中进行灵活调控。例如。通过调控打印挤出压力和打印的线条移动速度来获取所期望的尺寸大小。其中，所述生物墨水的固含量不同或粘度不同，对其均有影响；当所述生物墨水的粘度较大时，打印所需要的压力较大；粘度较小时，打印的压力也降低。打印的线条移动速度与直径(外径和内径)大小成反比，例如移动速度越大，线条越细；反之亦然。
可选地，所述小口径管状结构支架半成品的内径为0.1-2.8mm，所述小口径管状结构支架的外径为0.5-6mm。
可选地，所述3D同轴打印工艺中，使用80-200kPa的挤出压力将所述小口径管状结构支架从3D打印装置中挤出。一实施方式中，采用80-150kPa的挤出压力将所述小口径管状结构支架从3D打印装置中挤出。
可选地，所述光照射交联过程中，使用紫外光照射，所述紫外光的中心波长为360-370nm，交联时间为0.25-60min。例如使用UV交联剂装置进行紫外交联。所述交联时间还可以为1-20min。基于不同强度的紫外光，其交联时间进行调节。
本申请实施方式中，将所述得到小口径管状结构支架放入缓冲溶液中浸泡，待所述小口径管状结构支架溶胀平衡后，对溶胀平衡后的所述小口径管状结构支架进行性能测试。可选地，所述缓冲液可以但不限于为PBS缓冲液。本申请中，经溶胀平衡后的小口径管状结构支架能够使性能测试数据更可靠。
本申请第三方面所述的制备方法，能够一步式制备兼具尺寸可控性和力学可调节性的高强度小口径管状结构支架；该制备方法制作方法简单，成本低，可适用于工业化生产。
第四方面，本申请还提供看一种如本申请第二方面所述的小口径管状结构支架或本申请第三方面所述制备方法制备的小口径管状结构支架在人造组织、药物筛选和病理模型中的应用。
本申请所述小口径管状结构尺寸可控，且所述小口径管状结构具有突出的机械性能和良好生物相容性，能用以模拟人体尿管、肠管、食管、气管、胆管、血管等管状组织，在人造组织、药物筛选和病理模型研究中具有广泛应用前景。
由于所述小口径管状结构可以做到尺寸更小的同时，仍保持突出的机械性能，因此，所述小口径管状结构尤其具有突出的机械性能，在生物医疗领域或组织工程领域具有广泛应用。例如，所述小口径管状结构可以用于解决人体组织器官移植供体来源缺乏、免疫排斥的问题。
本申请的优点将会在下面的说明书中部分阐明，一部分根据说明书是显而易见的，或者可以通过本申请实施例的实施而获知。
附图说明
为更清楚地阐述本申请的内容，下面结合附图与具体实施例来对其进行详细说明。
图1为本申请一实施例提供的NAGA单体的氢谱谱图；
图2为本申请一实施例提供的明胶和GelMA的氢谱谱图；
图3为本申请一实施例提供的CNG生物墨水的红外光谱图；
图4为本申请一实施例提供的小口径管状结构支架的3D打印制备示意图；
图5为本申请一实施例提供的不同直径的小口径管状结构支架实际样品图；
图6为本申请一实施例提供的小口径管状结构支架的扫描电镜图；
图7为本申请一实施例提供的小口径管状结构支架的拉伸性能表征图；
图8为本申请一实施例提供的小口径管状结构支架的爆破压和断裂缝合测试图；
图9为本申请一实施例提供的小口径管状结构支架的抗疲劳性能测试图。
具体实施方式
以下所述是本申请实施例的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请实施例原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本申请实施例的保护范围。
本申请说明书、权利要求书和附图中出现的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤或单元的过程、方法、系统、产品或设备没有限定于已列出的步骤或单元，而是可选地还包括没有列出的步骤或单元，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤或单元。
若无特别说明，本申请实施例所采用的原料及其它化学试剂皆为市售商品。
本申请一实施例提供了一种小口径管状结构支架的制备方法。
(1)N-丙烯酰基甘氨酰胺(NAGA)的制备
以甘氨酰胺盐酸盐和丙烯酰氯为原材料制备获得，利用核磁共振 1H NMR(NMR，500MHz，瓦里安伊诺瓦(Varian INOVA))证明了其成功合成，参见图1，其中结构式中的a、b和c分别为碳原子上的氢，其特征峰如下： 1H NMR(D 2O)：δ＝4.1(H c，-NH-CH 2-)，5.6(H a，CH 2＝CH-)，6.0和6.1(H b，CH 2＝CH-)ppm。
(2)改性明胶(GelMA)的制备
以猪皮来源的明胶和甲基丙烯酸酐为原料。
首先，将猪皮明胶以10％(w/v)的比例混到PBS缓冲液中，并于50℃水浴加热下搅拌直至其成分完全溶解。随后，向明胶溶液中逐滴添加8mL(v/v)的甲基丙烯酸酐，并在50℃下反应3小时形成GelMA溶液。然后将溶液稀释并通过在40℃下使用分子量为12-14kDa透析袋中用蒸馏水进行透析一周，去除溶液中小分子量的产物及反应物；将透析后得到的改性明胶溶液冻干4天，产生白色多孔泡沫状产物，然后将产生的泡沫状产物储存在-80℃以备用。
参见图2，图2为明胶和改性明胶(GelMA)的核磁共振氢谱谱图。通过两个谱图对比发现，GelMA的图谱在&＝5.65和&＝5.33的位置上有明显的两个峰，即为赖氨酸基团以及羟基赖氨酸基团上甲基丙烯酰胺的双键的质子峰，这说明双键已经成功接到明胶分子链上，即GelMA成功合成。另外，通过 1H NMR(NMR，500MHz，瓦里安伊诺瓦(Varian INOVA))测定GelMA双键接枝率。明胶和改性明胶以浓度为10mg/mL溶解于D 2O中，结果显示改性明胶中的双键接枝率为81％左右。
(3)生物墨水的合成
先将GelMA以一定浓度溶解于去离子水中，于50℃-60℃水浴充分溶解，然后再把NAGA单体和Clay颗粒按照一定比例与一定浓度GelMA溶液充分混 匀，形成NAGA/Clay/GelMA水凝胶体系(简称CNG)，然后在CNG中加入适量光引发剂，避光搅拌至引发剂完全溶解，得到CNG生物墨水。其中，NAGA/GelMA的质量比可以为1:9，3:7，5:5，7:3，9:1。所述NAGA、GelMA、Clay和光引发剂在CNG生物墨水的质量含量可以分别为10％-30％、1％-16％、3％-10％和0.1％-0.5％。
取部分制备的CNG生物墨水注入到密封的模具夹板中，将其放入紫外交联仪中，在特定紫外强度(中心波长为365nm)下交联特定时间(0-60min)。充分交联后，取出凝胶并将其浸泡在PBS中过夜，达到溶胀平衡，即得到CNG高强度水凝胶，然后进行红外检测。参见图3，图3为CNG生物墨水的红外光谱图。图中，NAGA水凝胶还显示了3365、3194和3084cm -1处的NH伸缩振动峰，1546cm -1处的NH变形振动峰和1662cm -1处的C＝O伸缩振动峰(A)。同时，GelMA的特征峰显示明胶主链的特征峰(B)。在3165cm -1和3073cm -1处的强吸收峰归因于NH波段的伸缩振动(酰胺A)，在1662cm -1处的特征峰归因于C＝O的伸缩振动(酰胺I)。在1540cm -1处的特征带归因于NH带(酰胺II)的拉伸振动，在1250cm -1处的特征峰归因于氨基酸侧链(酰胺III)的CN拉伸振动。同时，在Clay/NAGA/GelMA水凝胶的光谱中，除了出现NAGA和GelMA的特征峰外也有纳米粘土(Clay)的特征峰。其中Si-O伸缩振动和弯曲振动峰出现在1006cm -1和660cm -1处。这结果表明NAGA/GelMA/Clay混合水凝胶成功交联。
(4)小口径管状结构支架的制备
参见图4，将上述制备的CNG生物墨水填着在物料筒中，通过3D打印装置(例如3D同轴打印机)，在压力为80-200kpa下，挤压出尺寸可控的小口径管状结构支架半成品，最后将其放入紫外交联仪中，在特定紫外强度(中心波 长为365nm)下交联特定时间(0.25-60min)，充分交联后，得到小口径管状结构支架。其中，纳米黏土颗粒可以起到物理穿插和增稠效果，既通过物理穿插方式，又通过化学交联方式提高小口径管状结构支架力学稳定性及生物活性。
下面分多个实施例对本发明实施例进行进一步的说明。
实施例1：一种小口径管状结构支架的制备方法，包括如下步骤：
将40mg的GelMA溶解于1mL的去离子水中并于50℃-60℃水浴充分溶解；
然后将100mg的黏土、360mg NAGA单体与GelMA混合溶液充分混匀；加入约3-6μL的紫外光引发剂1713，避光搅拌至引发剂完全溶解；获得Clay/NAGA/GelMA混合水凝胶生物墨水，简称CNG-01(其中，Clay的质量为100mg，NAGA/GelMA的质量比为9:1)。
将CNG-01混合水凝胶生物墨水装到打印物料筒中，通过3D同轴打印机，在压力为80-100kPa下，挤压出尺寸可控的空心管状结构(外直径OD为0.5mm-3mm，内直径ID为0.1-2.8mm)，最后将其放入紫外交联仪中，在中心波长为365nm的特定紫外强度下交联40min，得到小口径管状结构支架样品。
实施例2：一种小口径管状结构支架的制备方法，包括如下步骤：
将120mg的GelMA溶解于1mL的去离子水中并于50℃-60℃水浴充分溶解；
然后将100mg的黏土和280mg的NAGA单体与GelMA溶液充分混匀；加入约3-6μL的紫外光引发剂1713，避光搅拌至引发剂完全溶解；获得Clay/NAGA/GelMA混合水凝胶生物墨水，简称CNG-02(其中，Clay的质量为100mg，NAGA/GelMA的质量比为7:3)。
将CNG-02混合水凝胶生物墨水装到打印物料筒中，通过3D同轴打印机，在压力为100-120kPa下，挤压出尺寸可控的空心管状结构(外直径OD为 0.5mm-3mm，内直径ID为0.1-2.8mm)，最后将其放入紫外交联仪中，在中心波长为365nm的特定紫外强度下交联40min，得到小口径管状结构支架样品。
实施例3：一种小口径管状结构支架的制备方法，包括如下步骤：
将200mg的GelMA溶解于1mL的去离子水中并于50℃-60℃水浴充分溶解；
然后将100mg的黏土和200mg的NAGA单体与GelMA溶液充分混匀；加入约3-6μL的紫外光引发剂1713，避光搅拌至引发剂完全溶解；获得Clay/NAGA/GelMA混合水凝胶生物墨水，简称CNG-03(其中，Clay的质量为100mg，NAGA/GelMA的质量比为5:5)。
将CNG-03混合水凝胶生物墨水装到打印物料筒中，通过3D同轴打印机，在压力为100-130kPa下，挤压出尺寸可控的空心管状结构(外直径OD为0.5mm-3mm，内直径ID为0.1-2.8mm)，最后将其放入紫外交联仪中，在中心波长为365nm的特定紫外强度下交联40min，得到小口径管状结构支架样品。
实施例4：一种小口径管状结构支架的制备方法，包括如下步骤：
将200mg双键修饰的透明质酸(Hyaluronan，缩写HA)溶解于1mL的去离子水中充分溶解；
然后将100mg的黏土和200mg的NAGA单体与透明质酸溶液充分混匀；加入约3-6μL的紫外光引发剂1713，避光搅拌至引发剂完全溶解；获得Clay/NAGA/双键修饰的HA的混合水凝胶生物墨水，简称CNH-01(其中，Clay的质量为100mg，NAGA与双键修饰的HA的质量比为5:5)。
将CNH-01混合水凝胶生物墨水装到打印物料筒中，通过3D同轴打印机，挤压出尺寸可控的空心管状结构(外直径OD为0.5mm-6mm，内直径ID为0.1-2.8mm)，最后将其放入紫外交联仪中，在中心波长为365nm的特定紫外强度下交联40min，得到小口径管状结构支架样品。
实施例5：一种小口径管状结构支架的制备方法，包括如下步骤：
将200mg双键修饰的胶原溶解于1mL的去离子水中充分溶解；
然后将100mg的黏土和200mg的NAGA单体与双键修饰的胶原溶液充分混匀；加入约3-6μL的紫外光引发剂1713，避光搅拌至引发剂完全溶解；获得Clay/NAGA/双键修饰的胶原的混合水凝胶生物墨水，其中，Clay的质量为100mg，NAGA与双键修饰的胶原的质量比为5:5)。
将混合水凝胶生物墨水装到打印物料筒中，通过3D同轴打印机，挤压出尺寸可控的空心管状结构(外直径OD为0.5mm-6mm，内直径ID为0.1-2.8mm)，最后将其放入紫外交联仪中，在中心波长为365nm的特定紫外强度下交联40min，得到小口径管状结构支架样品。
实施例6：一种小口径管状结构支架的制备方法，包括如下步骤：
将200mg双键修饰的海藻酸钠溶解于1mL的去离子水中充分溶解；
然后将100mg的黏土和200mg的NAGA单体与双键修饰的海藻酸钠溶液充分混匀；加入约3-6μL的紫外光引发剂1713，避光搅拌至引发剂完全溶解；获得Clay/NAGA/双键修饰的海藻酸钠的混合水凝胶生物墨水，其中，Clay的质量为100mg，NAGA与双键修饰的海藻酸钠的质量比为5:5)。
将混合水凝胶生物墨水装到打印物料筒中，通过3D同轴打印机，挤压出尺寸可控的空心管状结构(外直径OD为0.5mm-6mm，内直径ID为0.1-2.8mm)，最后将其放入紫外交联仪中，在中心波长为365nm的特定紫外强度下交联40min，得到小口径管状结构支架样品。
效果实施例：
(1)不同直径的小口径管状结构支架的制备
基于本申请实施例所述制备方法，制备不同直径的小口径管状结构支架样品，参见图5中A至图5中F，A-F中小口径管状结构支架的外径D分别为：3mm，2.8mm，2.0mm，1.5mm，1.2mm和0.8mm。随机抽取一个小口径管状结构支架样品进行扫描电镜测试，结果如图6所述。
从图中的呈现的不同直径的小口径管状结构支架样品数据可知，本申请所述制备方法可以用于制备不同直径(外直径OD为0.5mm-3mm，内直径ID为0.1-2.8mm)的小口径管状结构支架，制得的小口径管状结构支架结构稳定。
(2)小口径管状结构支架的性能测定
取上述实施例1-3制得的小口径管状结构支架，采用如下方法测试各实施例的小口径管状结构支架的力学性能：
(a)拉伸性能：在电子万能试验机上进行，拉伸的样品直径3mm(外径/内径约为3mm/2.4mm)，拉伸速率为50mm/min，如图7所示，分别对CNG-01(组别a)、CNG-02(组别b)、CNG-03(组别c)、生物食道管(组别d)和主动脉管(组别e)。
结果显示，实施例1制备得到的小口径管状结构支架CNG-01，在外径/内径约为3mm/2.4mm下，拉伸断裂强度(～22MPa)，拉伸率(～500％)；实施例2制备得到的小口径管状结构支架CNG-02的拉伸断裂强度(～10MPa)，拉伸率(～60％)；实施例3制备得到的小口径管状结构支架CNG-03拉伸断裂强度(～8MPa)，拉伸率(～43％)。从性能测试结果数据可知，本申请实施例制备得到小口径管状结构支架具有43-500％的拉伸率，机械性能出色。同时对各个实施例的杨氏模量进行测试，结果也显示实施例1-3的杨氏模量分别可达35MPa、24MP和21MPa。
(b)爆破压和断裂缝合测试：爆破压力试验按国标AS ISO7198-2003进行。 通过自制的抗爆破压装置平台进行检测管状结构支架抗爆破性能。总长为4cm，直径为3mm(外径/内径约为3mm/2.4mm)的测试样品，用6-0丝线把空心管固定到装置上，以50mmHg s -1的速率增加压力，直到微管破裂。记录管状结构支架破裂时的破裂压力(mmHg)。按照ASISO 7198-2003进行断裂缝合试验。小口径管状结构支架样品总长度约为3cm。把管状结构支架的一端固定到单轴拉伸试验机的台夹上，在距离边缘3mm处，用6～0聚丙烯缝线针穿过管子的管壁，以50mm/min的恒定速率拉动缝合线，直到试样完全撕裂；最大的力被记录为缝合保持强度。取实施例1和实施例2制备的各个外径/内径的小口径管状结构支架进行测试，测试前，各组样品在37℃的PBS中浸泡过夜，结果如图8所示。
图8中A，组别4-6分别是实施例2中NAGA/GelMA的质量比7:3的小口径管状结构支架样品，外径/内径分别为：2.2mm/0.3mm、2.8mm/0.1mm、3mm/0.5mm。组别7-10分别是实施例1中NAGA/GelMA的质量比9:1的小口径管状结构支架样品，外径/内径分别为：2.2mm/0.3mm、2.2mm/0.3mm、2.8mm/0.1mm、3mm/0.3mm。图8中B，组别2-4分别是实施例2中NAGA/GelMA的质量比7:3的小口径管状结构支架样品，外径/内径分别为：2.2mm/0.3mm、2.8mm/0.1mm、3mm/0.5mm；组别5-8分别是实施例1中NAGA/GelMA的质量比9:1的小口径管状结构支架样品，外径/内径分别为：2.2mm/0.3mm、2.2mm/0.3mm、2.8mm/0.1mm、3mm/0.3mm。
从图8数据可知，本申请实施例1的小口径管状结构支架样品的爆破压可达约1900mmHg，缝合线保持强度可为280gf。实施例2的小口径管状结构支架样品的爆破压可达约2500mmHg，缝合线保持强度可为85gf。本申请实施例制备的小口径管状结构支架样品的抗爆破性能出色，缝合线保持强度高，其爆破压可以媲美，甚至优于现有的主动脉管、食道管和气管参数；其缝合线保持强 度也能够达到280gf，表现出出色的结构稳定性和机械性能，。
(c)抗疲劳性能测试，参见图9，为实施例1的小口径管状结构支架抗疲劳性能测试数据图，其中，其外径/内径约为3mm/2.4mm，所述小口径管状结构支架可承受500次以上的循环应力测试。
需要说明的是，根据上述说明书的揭示和阐述，本申请所属领域的技术人员还可以对上述实施方式进行变更和修改。因此，本申请并不局限于上面揭示和描述的具体实施方式，对本申请的一些等同修改和变更也应当在本申请的权利要求的保护范围之内。此外，尽管本说明书中使用了一些特定的术语，但这些术语只是为了方便说明，并不对本申请构成任何限制。

Claims (20)

  1. 一种生物墨水，用于3D打印，其特征在于，包括N-丙烯酰基甘氨酰胺、高分子聚合物和纳米黏土，其中，所述高分子聚合物包括改性明胶、双键修饰的海藻酸盐、双键修饰的胶原和双键修饰的透明质酸中的一种或多种；所述N-丙烯酰基甘氨酰胺和所述高分子聚合物的质量比为(0.1-10):1。
  2. 如权利要求1所述的生物墨水，其特征在于，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量为10％-30％。
  3. 如权利要求1所述的生物墨水，其特征在于，所述改性明胶为甲基丙烯酸酐改性明胶；所述改性明胶的双键接枝率大于70％。
  4. 如权利要求1所述的生物墨水，其特征在于，所述生物墨水还包括光引发剂，所述光引发剂在所述生物墨水中的质量百分含量为0.1％-0.5％。
  5. 如权利要求1所述的生物墨水，其特征在于，所述高分子聚合物在所述生物墨水中的质量百分含量为1％-16％。
  6. 如权利要求1所述的生物墨水，其特征在于，所述纳米黏土为纳米片状，所述纳米黏土的横向尺寸为20-40nm；厚度为0.5-5nm。
  7. 如权利要求5所述的生物墨水，其特征在于，所述纳米黏土在所述生物墨水中的质量百分含量为3％-10％。
  8. 一种小口径管状结构支架，其特征在于，所述小口径管状结构支架由权利要求1-7任一项所述生物墨水经3D打印制备得到。
  9. 如权利要求8所述的小口径管状结构支架，其特征在于，所述小口径管状结构支架包括至少一根空心管状结构，所述空心管状结构的管壁包括单层或多层复合材料层，所述复合材料层由所述生物墨水固化形成。
  10. 如权利要求9所述的小口径管状结构支架，其特征在于，所述空心管状结构的管壁包括从内向外依次层叠的内层、中间层和外层复合材料层。
  11. 如权利要求9或10所述的小口径管状结构支架，其特征在于，所述小口径管状结构支架的内径为0.1-2.8mm，所述小口径管状结构支架的外径为0.5-6.0mm。
  12. 如权利要求9或10所述的小口径管状结构支架，其特征在于，所述小口径管状结构支架的内径为0.1-2.0mm，所述小口径管状结构支架的外径为0.5-3.0mm。
  13. 如权利要求8-12任一项所述的小口径管状结构支架，其特征在于，所述小口径管状结构支架的拉伸断裂强度为10-30MPa；所述小口径管状结构支架的拉伸率为40-500％。
  14. 如权利要求8-12任一项所述的小口径管状结构支架，其特征在于，所述小口径管状结构支架的缝合线保持强度为80-300克力。
  15. 一种小口径管状结构支架的制备方法，其特征在于，包括以下步骤：
    配制高分子聚合物水溶液，加入N-丙烯酰基甘氨酰胺和纳米黏土，混合均匀后得到混合料，所述高分子聚合物包括改性明胶、双键修饰的海藻酸盐、双键修饰的胶原和双键修饰的透明质酸中的一种或多种，所述N-丙烯酰基甘氨酰胺和所述高分子聚合物的质量比为(0.1-10):1；
    向所述混合料中加入光引发剂，避光搅拌均匀后，得到生物墨水，将所述生物墨水填装至打印物料筒中，采用3D同轴打印工艺按预设尺寸进行打印，然后经光照射交联固化后，得到小口径管状结构支架。
  16. 如权利要求15所述的制备方法，其特征在于，所述N-丙烯酰基甘氨酰胺在所述生物墨水中的质量百分含量为10％-30％。
  17. 如权利要求15所述的制备方法，其特征在于，所述改性明胶为甲基丙烯酸酐改性明胶；所述改性明胶的双键接枝率大于70％。
  18. 如权利要求15所述的制备方法，其特征在于，所述纳米黏土在所述生物墨水中的质量百分含量为3％-10％；所述纳米黏土为纳米片状，所述纳米黏土的横向尺寸为20-40nm；厚度为0.5-5nm。
  19. 如权利要求15所述的制备方法，其特征在于，所述小口径管状结构支架半成品的内径为0.1-2.8mm，所述小口径管状结构支架的外径为0.5-6mm。
  20. 一种如权利要求8-14任一项所述的小口径管状结构支架或如权利要求15-19任一项所述制备方法制备的小口径管状结构支架在人造组织、药物筛选和病理模型中的应用。
PCT/CN2020/088427 2020-04-30 2020-04-30 生物墨水、小口径管状结构支架及其制备方法和应用 WO2021217621A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/088427 WO2021217621A1 (zh) 2020-04-30 2020-04-30 生物墨水、小口径管状结构支架及其制备方法和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/088427 WO2021217621A1 (zh) 2020-04-30 2020-04-30 生物墨水、小口径管状结构支架及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2021217621A1 true WO2021217621A1 (zh) 2021-11-04

Family

ID=78331638

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/088427 WO2021217621A1 (zh) 2020-04-30 2020-04-30 生物墨水、小口径管状结构支架及其制备方法和应用

Country Status (1)

Country Link
WO (1) WO2021217621A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109206634A (zh) * 2017-06-30 2019-01-15 天津大学 明胶基高强度水凝胶及其制备方法
WO2019129560A1 (en) * 2017-12-29 2019-07-04 Basf Se A composition to produce support sub-structures for 3d photopolymer jetting
CN110655621A (zh) * 2019-09-25 2020-01-07 中国科学院深圳先进技术研究院 复合水凝胶及制备方法和应用
CN110885524A (zh) * 2018-09-11 2020-03-17 天津大学 基于聚丙烯酰基甘氨酰胺和海藻酸钠的双网络水凝胶及其制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109206634A (zh) * 2017-06-30 2019-01-15 天津大学 明胶基高强度水凝胶及其制备方法
WO2019129560A1 (en) * 2017-12-29 2019-07-04 Basf Se A composition to produce support sub-structures for 3d photopolymer jetting
CN110885524A (zh) * 2018-09-11 2020-03-17 天津大学 基于聚丙烯酰基甘氨酰胺和海藻酸钠的双网络水凝胶及其制备方法
CN110655621A (zh) * 2019-09-25 2020-01-07 中国科学院深圳先进技术研究院 复合水凝胶及制备方法和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AFEWERKI SAMSON, MAGALHÃES LEILA S. S. M., SILVA ANDRÉ D. R., STOCCO THIAGO D., SILVA FILHO EDSON C., MARCIANO FERNANDA R., LOBO A: "Bioprinting a Synthetic Smectic Clay for Orthopedic Applications", ADVANCED HEALTHCARE MATERIALS, vol. 8, no. 13, 31 July 2019 (2019-07-31), DE, pages 1 - 14, XP055861980, ISSN: 2192-2640, DOI: 10.1002/adhm.201900158 *
FEI GAO , CHANGSHUN RUAN , WENGUANG LIU: "High-strength Hydrogel-based Bioinks", MATERIALS CHEMISTRY FRONTIERS, vol. 3, no. 9, 22 August 2019 (2019-08-22), pages 1736 - 1746, XP055861972, DOI: 10.1039/C9QM00373H *
GAO FEI, XU ZIYANG, LIANG QINGFEI, LI HAOFEI, PENG LIUQI, WU MINGMING, ZHAO XIAOLI, CUI XU, RUAN CHANGSHUN, LIU WENGUANG: "Osteochondral Regeneration with 3d-printed Biodegradable High-Strength Supramolecular Polymer Reinforced-Gelatin Hydrogel Scaffolds", ADVANCED SCIENCE, vol. 6, no. 15, 31 August 2019 (2019-08-31), pages 1 - 12, XP055861986, ISSN: 2198-3844, DOI: 10.1002/advs.201900867 *
ZHAI XINYUN, MA YUFEI, HOU CHUNYONG, GAO FEI, ZHANG YINYU, RUAN CHANGSHUN, PAN HAOBO, LU WILLIAM WEIJIA, LIU WENGUANG: "3D-Printed High Strength Bioactive Supramolecular Polymer/Clay Nanocomposite Hydrogel Scaffold for Bone Regeneration", ACS BIOMATERIALS SCIENCE & ENGINEERING, vol. 3, no. 6, 12 June 2017 (2017-06-12), US, pages 1109 - 1118, XP055861973, ISSN: 2373-9878, DOI: 10.1021/acsbiomaterials.7b00224 *

Similar Documents

Publication Publication Date Title
CN113713179B (zh) 高综合性能光固化生物3d打印复合水凝胶及其制备方法和应用
US20240100224A1 (en) Two-field coupling crosslinked, injectable, moldable and printable granular hydrogel material, preparation method thereof and application thereof
CN111388750B (zh) 生物墨水、小口径管状结构支架及其制备方法和应用
CN113679888B (zh) 光固化成型复合水凝胶基质前驱体及其制备方法和带有其的支架
CN112126080B (zh) 基于巯基-烯点击反应的光固化水凝胶、其制法与应用
CN114874455B (zh) 一种中性溶解、具有自组装能力和光交联能力的改性胶原和凝胶的构建方法
CN113150561B (zh) 一种用于3d生物打印的胶原基生物墨水及其制备方法与应用
US9327054B2 (en) Nerve guide conduit containing carbon nanotubes
CN112812324A (zh) 一种聚阳离子-透明质酸复合水凝胶及其制备方法与应用
CN113975462B (zh) 一种多巴胺功能化的神经活性水凝胶的制备方法
Zhou et al. A 3D printing mold method for rapid fabrication of artificial blood vessels
WO2021217621A1 (zh) 生物墨水、小口径管状结构支架及其制备方法和应用
WO2024109011A1 (zh) 一种具有定向排列纤维微结构的高强度互穿网络水凝胶血管的制备方法
CN109758608B (zh) 具有高韧性的可打印复合水凝胶及制备方法与应用
CN115068700B (zh) 一种多功能复合腹壁疝气修复补片及其制备方法
CN113855854B (zh) 4d构建性能的生物材料及其制备方法和应用
TWI319980B (en) Laminin-modified conduit for nerve regeneration and methods of manufacturing the conduit and regenerating nerves using the conduit
CN115779147A (zh) 兼顾良好力学性能和高细胞增殖能力的双网络水凝胶用于制备生物组织工程支架的方法
CN111068108B (zh) 一种用于修复四肢长管骨骨缺损的骨移植支架
CN116173287B (zh) 一种用于膀胱缺损修复的水凝胶粘合剂、其制法和应用
CN109646716B (zh) 人工角膜光学中心部及其制备方法和人工角膜
WO2024078310A1 (zh) 一种胶原水凝胶及其制备方法和应用
US20230405187A1 (en) Composite hydrogel for light-cured 3d cell-laden printing and preparation method and application thereof
CN115501398B (zh) 一种生物相容性抗炎水凝胶涂层及其制备方法
CN112126069B (zh) 基于胶原和透明质酸的可快速固化水凝胶、其制法及应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20933506

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20933506

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205 DATED 20/04/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 20933506

Country of ref document: EP

Kind code of ref document: A1