CN119318736A - Preparation method and application of gel biological ink for 3D printing of small-diameter artificial blood vessels - Google Patents
Preparation method and application of gel biological ink for 3D printing of small-diameter artificial blood vessels Download PDFInfo
- Publication number
- CN119318736A CN119318736A CN202411444696.7A CN202411444696A CN119318736A CN 119318736 A CN119318736 A CN 119318736A CN 202411444696 A CN202411444696 A CN 202411444696A CN 119318736 A CN119318736 A CN 119318736A
- Authority
- CN
- China
- Prior art keywords
- printing
- ink
- gel
- artificial blood
- solution
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 210000004204 blood vessel Anatomy 0.000 title claims abstract description 71
- 238000010146 3D printing Methods 0.000 title claims abstract description 64
- 239000002473 artificial blood Substances 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- 102000008186 Collagen Human genes 0.000 claims abstract description 83
- 108010035532 Collagen Proteins 0.000 claims abstract description 83
- 229920001436 collagen Polymers 0.000 claims abstract description 83
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 58
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 58
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 48
- 230000002792 vascular Effects 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 53
- -1 methacryloyl Chemical group 0.000 claims description 32
- 239000008367 deionised water Substances 0.000 claims description 31
- 229910021641 deionized water Inorganic materials 0.000 claims description 31
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims description 29
- 238000003756 stirring Methods 0.000 claims description 29
- 238000007639 printing Methods 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 19
- 229920002385 Sodium hyaluronate Polymers 0.000 claims description 15
- 238000004108 freeze drying Methods 0.000 claims description 15
- 229940010747 sodium hyaluronate Drugs 0.000 claims description 15
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims description 15
- 238000001125 extrusion Methods 0.000 claims description 10
- 230000001678 irradiating effect Effects 0.000 claims description 10
- 238000004132 cross linking Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- RKCKOWJWQLITLM-UHFFFAOYSA-N P(O)(O)=O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C Chemical compound P(O)(O)=O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C RKCKOWJWQLITLM-UHFFFAOYSA-N 0.000 claims description 6
- 238000011049 filling Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 abstract description 8
- 239000008280 blood Substances 0.000 abstract description 8
- 208000037803 restenosis Diseases 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000000016 photochemical curing Methods 0.000 abstract 2
- 239000000243 solution Substances 0.000 description 88
- 239000000499 gel Substances 0.000 description 75
- 239000000017 hydrogel Substances 0.000 description 27
- 238000010586 diagram Methods 0.000 description 13
- 239000000047 product Substances 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 210000002889 endothelial cell Anatomy 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000002386 leaching Methods 0.000 description 5
- 230000005012 migration Effects 0.000 description 5
- 238000013508 migration Methods 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 5
- 238000010998 test method Methods 0.000 description 5
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 230000012292 cell migration Effects 0.000 description 4
- 210000002744 extracellular matrix Anatomy 0.000 description 4
- 238000010571 fourier transform-infrared absorption spectrum Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241000672609 Escherichia coli BL21 Species 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 3
- 229910002091 carbon monoxide Inorganic materials 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000002902 bimodal effect Effects 0.000 description 2
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012876 topography Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000031481 Pathologic Constriction Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 206010053648 Vascular occlusion Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000004177 elastic tissue Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 208000037804 stenosis Diseases 0.000 description 1
- 230000036262 stenosis Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 208000021331 vascular occlusion disease Diseases 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
- A61F2/06—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
- 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
- A61L27/50—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
- 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
- A61L27/52—Hydrogels or hydrocolloids
-
- 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
- B33Y70/10—Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Engineering & Computer Science (AREA)
- Vascular Medicine (AREA)
- Biomedical Technology (AREA)
- Heart & Thoracic Surgery (AREA)
- Ceramic Engineering (AREA)
- Civil Engineering (AREA)
- Composite Materials (AREA)
- Structural Engineering (AREA)
- Manufacturing & Machinery (AREA)
- Materials Engineering (AREA)
- Cardiology (AREA)
- Pulmonology (AREA)
- Gastroenterology & Hepatology (AREA)
- Dispersion Chemistry (AREA)
- Biophysics (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明公开了一种3D打印小直径人工血管所用凝胶生物墨水的制备方法和应用，包括：提供甲基丙烯酰化重组胶原蛋白；提供甲基丙烯酰化透明质酸；以甲基丙烯酰化重组胶原蛋白、甲基丙烯酰化透明质酸为原料获得制备3D打印小直径人工血管所用凝胶生物墨水。该凝胶生物墨水具有较强的光固化特性且具备生物打印所需的剪切稀化特性，通过光固化3D打印技术可以成功制备得到直径为6mm，高为1cm人工血管支架，该支符合小直径血管的尺寸要求。利用本发明的凝胶生物墨水打印的血管支架血液相容性能良好，能有效的促架实现快速内皮化从而预防血管再狭窄等优点，是未来小口径血管替代物的潜在候选者，在人工血管市场上具备广阔的应用前景。
The present invention discloses a preparation method and application of gel bio-ink used for 3D printing of small-diameter artificial blood vessels, including: providing methacrylylated recombinant collagen; providing methacrylylated hyaluronic acid; using methacrylylated recombinant collagen and methacrylylated hyaluronic acid as raw materials to obtain the gel bio-ink used for preparing 3D printing of small-diameter artificial blood vessels. The gel bio-ink has strong photocuring properties and possesses the shear-thinning properties required for bioprinting. Through photocuring 3D printing technology, an artificial blood vessel stent with a diameter of 6 mm and a height of 1 cm can be successfully prepared, which meets the size requirements of small-diameter blood vessels. The blood vessel stent printed with the gel bio-ink of the present invention has good blood compatibility, can effectively promote the rapid endothelialization of the stent, thereby preventing vascular restenosis, etc. It is a potential candidate for small-caliber blood vessel substitutes in the future and has broad application prospects in the artificial blood vessel market.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a preparation method and application of gel biological ink for 3D printing of small-diameter artificial blood vessels.
Background
The blood vessel mainly comprises collagen fiber, elastic fiber, glycosaminoglycan and other macromolecular substances, and is used as a main channel of blood circulation to play important roles in providing oxygen and nutrient substances for various tissues and cells of a body, regulating body temperature, participating in immune response and the like. Vascular prostheses are considered as important substitutes for autografts in the treatment of cardiovascular diseases due to the limitations of autologous blood vessels. Large diameter vascular grafts (D >6 mm) have been used clinically, however, there are significant limitations in the use of small diameter vascular grafts (D <6 mm).
In the field of small-diameter vascular stents, restenosis is always a key difficulty in the research of small-caliber artificial blood vessels. Due to the lumen stenosis of small diameter vascular stents, the flow of blood therein is easily impeded, increasing the risk of thrombosis and vascular occlusion. The key to solving this problem is to achieve rapid endothelialization. Endothelial cells are important components of the inner wall of blood vessels and they are capable of secreting a variety of biologically active substances, maintaining normal function of blood vessels. By promoting rapid growth and coverage of endothelial cells on the surface of small diameter vascular stents, the occurrence of thrombosis and restenosis may be effectively reduced. How to achieve rapid endothelialization is critical to solve this problem.
3D printing is widely used in regenerative medicine and promotes significant innovation. In addition, in clinical treatment, due to the lack of ideal bionic natural vascular composition, the success rate of surgery and the rehabilitation effect of patients are often limited to a certain extent. Hydrogel materials with excellent biocompatibility have been the hot spot in the research field because of their physicochemical properties very similar to those of extracellular matrix.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of gel biological ink for 3D printing of small-diameter artificial blood vessels aiming at the defects of the prior art. The gel biological ink prepared by the invention has strong light curing property and shear thinning property required by biological printing, and the artificial vascular stent with the diameter of 6mm and the height of 1cm can be successfully prepared by a light curing 3D printing technology, thereby meeting the size requirement of small-diameter blood vessels (D <6 mm). The vascular stent has good blood compatibility, can effectively promote proliferation and migration of endothelial cells, and promote rapid endothelialization of the stent, thereby preventing restenosis of blood vessels.
In order to solve the technical problems, the technical scheme adopted by the invention is that the preparation method of the gel biological ink for 3D printing of the small-diameter artificial blood vessel is characterized by comprising the following steps:
Step one, dissolving methacryloyl recombinant collagen and a photoinitiator in deionized water under a light-shielding condition to obtain solution A;
Step two, mixing the solution A and the solution B in the step one to obtain gel solution;
and thirdly, standing the gel liquid in the second step in a refrigerator at the temperature of 4 ℃ for 8-36 hours to obtain the gel biological ink for 3D printing of the small-diameter artificial blood vessel.
The preparation method of the gel bio-ink for the 3D printing small-diameter artificial blood vessel is characterized by specifically comprising the steps of dropwise adding glycidyl methacrylate into a recombinant collagen solution, stirring and reacting for 6-8 hours in a 37 ℃ water bath, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen.
The preparation method of the gel bio-ink for the 3D printing small-diameter artificial blood vessel is characterized in that the recombinant collagen solution is obtained by dissolving recombinant collagen in deionized water, the mass-volume concentration of the recombinant collagen solution is 1% -2%, the relative molecular mass of the recombinant collagen is 97kDa, and the dosage of the glycidyl methacrylate is 10-30 mL of glycidyl methacrylate for each g of recombinant collagen.
The preparation method of the gel bio-ink for the 3D printing small-diameter artificial blood vessel is characterized by specifically comprising the steps of dropwise adding glycidyl methacrylate into a hyaluronic acid solution, stirring and reacting for 6-8 hours in a water bath at 60 ℃, dialyzing, and freeze-drying to obtain the methacryloyl hyaluronic acid.
The preparation method of the gel bio-ink for the 3D printing small-diameter artificial blood vessel is characterized in that the hyaluronic acid solution is obtained by dissolving sodium hyaluronate in deionized water, the mass-volume concentration of the hyaluronic acid solution is 1% -2%, the relative molecular mass of the sodium hyaluronate is 10 KDa-500 KDa, and the dosage of the glycidyl methacrylate is 10-30 mL of glycidyl methacrylate for every g of sodium hyaluronate.
The preparation method of the gel bio-ink for 3D printing of the small-diameter artificial blood vessel is characterized in that the photoinitiator in the first step is phenyl-2, 4, 6-trimethyl benzoyl lithium phosphonate.
The preparation method of the gel bio-ink for the 3D printing small-diameter artificial blood vessel is characterized in that the mass-volume concentration of the methacryloyl recombinant collagen in the gel liquid in the second step is 5% -10%, the mass-volume concentration of the methacryloyl hyaluronic acid is 1.5% -2%, and the mass-volume concentration of the photoinitiator is 0.1% -0.25%.
Furthermore, the invention also provides application of the gel bio-ink prepared by the method in preparation of a 3D printing bracket for preparing an artificial blood vessel.
The application is characterized in that the preparation method of the 3D printing bracket for preparing the artificial blood vessel comprises the steps of filling the gel bio-ink into a charging barrel of a 3D printer for 3D printing, contacting ultraviolet light after the gel bio-ink is extruded and irradiating for 3 min-5 min for photo-crosslinking curing, and sterilizing to obtain the 3D printing bracket for the artificial blood vessel.
The application is characterized in that the nozzle specification of the 3D printer is 23G-26G, the printing speed is 10 mm/s-30 mm/s, the printing temperature is 25 ℃, and the extrusion amount is 4% -8%.
Compared with the prior art, the invention has the following advantages:
1. The gel biological ink has strong light curing property and shear thinning property required by biological printing, and can be successfully prepared into an artificial vascular stent with the diameter of 6mm and the height of 1cm by a light curing 3D printing technology, thereby meeting the size requirement of small-diameter blood vessels (D <6 mm). The vascular stent has good blood compatibility, can effectively promote proliferation and migration of endothelial cells, and promote rapid endothelialization of the stent, thereby preventing restenosis of blood vessels.
2. The invention adopts the recombined collagen modified by the glycidyl methacrylate, has photosensitive property which can effectively promote the ultraviolet curing process of the biological ink to obtain the target 3D printing hydrogel bracket, and adopts the hyaluronic acid modified by the glycidyl methacrylate to fully exert the excellent rheological property, so that the biological ink has fluidity and mechanical stability, and is convenient for extrusion of the biological ink and molding of extrudate.
3. The invention can effectively realize rapid endothelialization by utilizing the unique properties and functions of recombinant collagen and hyaluronic acid. The recombinant collagen can provide a good scaffold structure for the growth of endothelial cells, has a unique three-dimensional network structure, can simulate the extracellular matrix environment of natural blood vessels, and creates proper conditions for the adhesion, migration and proliferation of the endothelial cells. Hyaluronic acid participates in the signal transmission process of cells in vascular tissues, plays a role in regulating the growth, migration, differentiation and the like of endothelial cells, and can promote endothelialization of artificial blood vessels. Hyaluronic acid and recombinant collagen are mutually matched to provide powerful support for the growth of endothelial cells and the endothelialization of blood vessels.
4. The recombinant collagen and hyaluronic acid can simulate collagen fibers and glycosaminoglycans in blood vessels and serve as scaffolds simulating natural extracellular matrixes (extracellular matrix, ECM), support cell growth and guide tissue regeneration.
5. The preparation method provided by the invention is reliable in principle and beneficial to popularization and application, and the prepared gel biological ink has great potential and wide application prospect in the artificial blood vessel market.
The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and the examples.
Drawings
FIG. 1 is a schematic flow chart of the preparation of methacryloylated recombinant collagen according to example 1 of the present invention.
FIG. 2 is a schematic diagram showing the gel formation of the methacryloylated recombinant collagen prepared in example 1 of the present invention after ultraviolet irradiation under the action of a photoinitiator.
FIG. 3 is a chart showing the Fourier transform infrared absorption spectrum analysis of the methacryloylated recombinant collagen prepared in example 1 of the present invention.
FIG. 4 shows the result of 1 H-NMR analysis of methacryloylated recombinant collagen prepared in example 1 of the present invention.
FIG. 5 is a schematic diagram showing the flow of the process for producing methacryloylated hyaluronic acid in example 1 of the invention.
FIG. 6 is a schematic diagram showing the gel formation of the methacryloylated hyaluronic acid prepared in example 1 of the present invention after irradiation with ultraviolet light under the action of a photoinitiator.
FIG. 7 is a chart showing the Fourier transform infrared absorption spectrum of the methacryloylated hyaluronic acid prepared in example 1 of the present invention.
FIG. 8 shows the result of 1 H-NMR analysis of methacryloylated hyaluronic acid prepared in example 1 of the invention.
FIG. 9 is a schematic diagram showing the results of the extrudability test of the scaffolds prepared in examples 1 and 2 of the present invention.
Fig. 10 is a schematic diagram showing the results of stability test of scaffolds prepared in examples 1 and 2 of the present invention.
FIG. 11 is a cross-sectional scanning electron microscope image of the stent A prepared in example 1 on a scale of 100. Mu.m.
FIG. 12 is a surface scanning electron micrograph of the stent A prepared in example 1 at a scale of 100. Mu.m.
Fig. 13 is a topography of a stent a prepared in example 1 of the present invention.
FIG. 14 shows the effect of the gel bio-ink prepared in example 1 of the present invention on the viability of endothelial cells in human umbilical vein.
FIG. 15 is a graph showing the area of the scratch area in the test for the cell migration promoting ability of the gel bio-ink prepared in example 1 of the present invention.
FIG. 16 is a photograph showing the cell migration promoting ability test of the gel bio-ink prepared in example 1 of the present invention.
FIG. 17 is a photograph showing the result of blood compatibility test of the gel bio-ink prepared in example 1 of the present invention.
FIG. 18 is a quantification of the blood compatibility test of the gel bio-ink prepared in example 1 of the present invention.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
The invention provides a preparation method of gel bio-ink for 3D printing of small-diameter artificial blood vessels, which comprises the following steps:
Step one, dissolving methacryloyl recombinant collagen and a photoinitiator in deionized water under a light-shielding condition to obtain solution A;
The preparation method of the methacryloyl recombinant collagen concretely comprises the steps of dropwise adding glycidyl methacrylate into a recombinant collagen solution, stirring in a 37 ℃ water bath for reaction for 6-8 hours, dialyzing, freeze-drying to obtain the methacryloyl recombinant collagen, wherein the recombinant collagen solution is obtained by dissolving recombinant collagen in deionized water, the mass-volume concentration of the recombinant collagen solution is 1% -2%, the relative molecular mass of the recombinant collagen is 97kDa, and the dosage of the glycidyl methacrylate is 10-30 mL of glycidyl methacrylate for each g of recombinant collagen;
The preparation method of the methacrylic acid acylated hyaluronic acid specifically comprises the steps of dropwise adding glycidyl methacrylate into a hyaluronic acid solution, stirring in a 60 ℃ water bath for reaction for 6-8 hours, dialyzing, freeze-drying to obtain the methacrylic acid acylated hyaluronic acid, wherein the hyaluronic acid solution is a hyaluronic acid solution obtained by dissolving sodium hyaluronate in deionized water, the mass-volume concentration of the hyaluronic acid solution is 1% -2%, the relative molecular mass of the sodium hyaluronate is10 KDa-500 KDa, and the dosage of the glycidyl methacrylate is 10-30 mL of the glycidyl methacrylate for every g of sodium hyaluronate;
the photoinitiator is phenyl-2, 4, 6-trimethyl benzoyl lithium phosphonate;
Step two, mixing the solution A and the solution B in the step one to obtain a gel solution, wherein the mass-volume concentration of the methacryloyl recombinant collagen in the gel solution is 5% -10%, the mass-volume concentration of the methacryloyl hyaluronic acid is 1.5% -2%, and the mass-volume concentration of the photoinitiator is 0.1% -0.25%;
and thirdly, standing the gel liquid in the second step in a refrigerator at the temperature of 4 ℃ for 8-36 hours to obtain the gel biological ink for 3D printing of the small-diameter artificial blood vessel.
The preparation method of the methacryloyl recombinant collagen specifically comprises the steps of dropwise adding glycidyl methacrylate into a recombinant collagen solution, stirring and reacting for 6-8 hours in a 37 ℃ water bath, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen.
The gel bio-ink prepared by the method can be used for preparing a 3D printing bracket for preparing an artificial blood vessel, and the specific method comprises the steps of filling the gel bio-ink into a charging barrel of a 3D printer for 3D printing, contacting ultraviolet light and irradiating for 3 min-5 min for photo-crosslinking curing after the gel bio-ink is extruded at the same time of printing, and sterilizing to obtain the 3D printing bracket for the artificial blood vessel, wherein the specification of a nozzle of the 3D printer is 23G-26G, the printing speed is 10 mm/s-30 mm/s, the printing temperature is 25 ℃, and the extrusion amount is 4% -8%.
The present invention will be specifically described with reference to examples, which are not intended to limit the present invention.
Example 1
The embodiment provides a preparation method of gel bio-ink for 3D printing of small-diameter artificial blood vessels and a method for preparing 3D printing stents of the artificial blood vessels by using the gel bio-ink, which specifically comprise the following steps:
step one, providing methacryloyl recombinant collagen, which specifically comprises the following steps:
Step 101, 1g of recombinant collagen is dissolved in 100mL of deionized water to obtain a recombinant collagen solution, wherein the recombinant collagen is mRNA of human collagen is reversely transcribed to generate cDNA and then expressed in escherichia coli BL21, the structure and the obtaining method can refer to patent application files with the patent number ZL01106757.8 and the patent name of human-like collagen and the production method thereof, and the relative molecular mass of the recombinant collagen is 97kDa;
step 102, dropwise adding 30mL of glycidyl methacrylate into the recombinant collagen solution, stirring in a 37 ℃ water bath for reaction for 6 hours, and collecting a product;
Step 103, continuously dialyzing the product in deionized water for 7 days, and freeze-drying to obtain the methacryloylated collagen, wherein the cutoff molecular weight of the dialysis bag is 3500Da;
step two, providing the methacryloylated hyaluronic acid, which specifically comprises the following steps:
step 201, 1g of sodium hyaluronate is dissolved in 100mL of deionized water to obtain a hyaluronic acid solution, wherein the relative molecular mass of the sodium hyaluronate is 10kDa;
step 202, dropwise adding 30mL of glycidyl methacrylate into the hyaluronic acid solution, stirring and reacting for 6 hours in a water bath at 60 ℃, and collecting a product;
Step 203, continuously dialyzing the product in deionized water for 7 days, and freeze-drying to obtain the methacryloyl hyaluronic acid, wherein the cutoff molecular weight of the dialysis bag is 3500Da;
step three, providing gel biological ink for 3D printing of small-diameter artificial blood vessels, which comprises the following steps:
step 301, placing 500mg of methacryloyl recombinant collagen and 12.5mg of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) in 2.5mL of deionized water, sealing with tinfoil to avoid light, and stirring at room temperature until the materials are completely dissolved to obtain solution A, wherein the stirring time at room temperature can be 12 hours;
Step 302, placing 100mg of methacryloyl hyaluronic acid in 2.5mL of deionized water, and stirring in a 37 ℃ water bath until the hyaluronic acid is completely dissolved to obtain solution B, wherein the stirring time in the water bath can be 2 hours;
step 303, mixing the solution A in the step 301 and the solution B in the step 302, and continuing stirring for 1h to obtain a gel solution, wherein the gel solution has no obvious fluidity and is milky;
Step 304, standing the gel solution in a refrigerator at 4 ℃ for about 12 hours to obtain gel biological ink, and standing the gel solution at 4 ℃ for about 12 hours to fully crosslink and defoam the gel solution;
The method comprises the steps of taking gel biological ink as a raw material to prepare a 3D printing support for preparing an artificial blood vessel, and specifically comprises the steps of filling the gel biological ink into a charging barrel of a 3D printer, performing 3D printing to obtain the 3D printing support, wherein the model of the 3D printer is FOODBOT-S2, extruding the biological ink while printing, contacting ultraviolet light (365 nm,10mW/cm 2) after the biological ink is extruded, irradiating for 3min for photo-crosslinking and curing, and irradiating and sterilizing Co60 to obtain the 3D printing support for preparing the artificial blood vessel, namely a support A, wherein the diameter of the support A is 6mm, and the height of the support A is 1cm;
The specification of the nozzle of the 3D printer is 25G;
the printing speed is 10mm/s;
The printing temperature is 25 ℃;
the extrusion amount was 5%.
Comparative example 1
The comparative example examines the influence factors of the bio-ink hydration of the gel solution, and the preparation method comprises the following steps:
step one, 500mg of the methacryloyl recombinant collagen described in the example 1 and 12.5mg of a photoinitiator LAP are placed in 2.5mL of deionized water, the silver is sealed to avoid light, and the mixture is stirred at room temperature until the mixture is completely dissolved, so that a solution A is obtained;
step two, placing 25mg of the methacryloylated hyaluronic acid in example 1 in 2.5mL of deionized water, and stirring in a 37 ℃ water bath until the hyaluronic acid is completely dissolved to obtain solution B;
Step three, uniformly mixing the solution A and the solution B to obtain a mixed system C;
And fourthly, placing the mixed system C in an ultraviolet curing box, irradiating for 0.5min under 365nm irradiation, and then performing Co60 irradiation sterilization to obtain the hydrogel.
The hydrogel in this comparative example did not have printability, probably because the fluidity of the mixed system C was too strong and it did not have the shear thinning property.
Example 2
The embodiment provides a preparation method of gel bio-ink for 3D printing of small-diameter artificial blood vessels and a method for preparing 3D printing stents of the artificial blood vessels by using the gel bio-ink, which specifically comprise the following steps:
step one, providing methacryloyl recombinant collagen, which specifically comprises the following steps:
Step 101, 1.5g of recombinant collagen is dissolved in 100mL of deionized water to obtain a recombinant collagen solution, wherein the recombinant collagen is mRNA of human collagen is subjected to reverse transcription to generate cDNA and then is expressed in escherichia coli BL21, the structure and the acquisition method can be referred to as patent application files with the patent number ZL01106757.8 and the patent name of human-like collagen and the production method thereof, and the relative molecular mass of the recombinant collagen is 97kDa;
step 102, dropwise adding 15mL of glycidyl methacrylate into the recombinant collagen solution, stirring and reacting for 7 hours in a 37 ℃ water bath, and collecting a product;
Step 103, continuously dialyzing the product in deionized water for 7 days, and freeze-drying to obtain the methacryloyl recombinant collagen, wherein the cutoff molecular weight of the dialysis bag is 3500Da;
step two, providing the methacryloylated hyaluronic acid, which specifically comprises the following steps:
step 201, 2g of sodium hyaluronate is dissolved in 100mL of deionized water to obtain a hyaluronic acid solution, wherein the relative molecular mass of the sodium hyaluronate is 200kDa;
step 202, dropwise adding 20mL of glycidyl methacrylate into the hyaluronic acid solution, stirring and reacting for 7 hours in a water bath at 60 ℃, and collecting a product;
Step 203, continuously dialyzing the product in deionized water for 7 days, and freeze-drying to obtain the methacryloyl hyaluronic acid, wherein the cutoff molecular weight of the dialysis bag is 3500Da;
step three, providing 3D printing biological ink for preparing artificial blood vessels, which specifically comprises the following steps:
step 301, placing 400mg of methacryloyl recombinant collagen and 10mg of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) in 2.5mL of deionized water, sealing with tinfoil to avoid light, and stirring at room temperature until the materials are completely dissolved to obtain solution A, wherein the stirring time at room temperature can be 12 hours;
Step 302, placing 80mg of methacryloyl hyaluronic acid in 2.5mL of deionized water, and stirring in a 37 ℃ water bath until the hyaluronic acid is completely dissolved to obtain solution B, wherein the stirring time in the water bath can be 2 hours;
step 303, mixing the solution A in the step 301 and the solution B in the step 302, and continuing stirring for 1h to obtain a gel solution, wherein the gel solution has no obvious fluidity and is milky;
Step 304, standing the gel solution in a refrigerator at 4 ℃ for about 12 hours to obtain gel biological ink, and standing the gel solution at 4 ℃ for about 12 hours to fully crosslink and defoam the gel solution;
The method comprises the steps of taking gel bio-ink as a raw material to prepare a 3D printing bracket for preparing an artificial blood vessel, and specifically comprises the steps of filling the gel bio-ink into a charging barrel of a 3D printer, performing 3D printing to obtain the 3D printing bracket, wherein the model of the 3D printer is FOODBOT-S2, extruding the gel bio-ink, contacting ultraviolet light (365 nm,10mW/cm < 2 >) and irradiating for 4min for photo-crosslinking and curing, and irradiating and sterilizing Co60 to obtain the 3D printing hydrogel bracket for preparing the artificial blood vessel, namely a bracket B, wherein the diameter of the bracket B is 6mm and the height of the bracket B is 1cm;
The nozzle specification of the 3D printer is 26G;
The printing speed is 20mm/s;
The printing temperature is 25 ℃;
The extrusion amount was 4%.
The properties of the 3D printed hydrogel scaffold for preparing vascular prostheses of this example were substantially identical to those of example 1.
Example 3
The embodiment provides a preparation method of gel bio-ink for 3D printing of small-diameter artificial blood vessels and a method for preparing 3D printing stents of the artificial blood vessels by using the gel bio-ink, which specifically comprise the following steps:
step one, providing methacryloyl recombinant collagen, which specifically comprises the following steps:
Step 101, 2g of recombinant collagen is dissolved in 100mL of deionized water to obtain a recombinant collagen solution, wherein the recombinant collagen is mRNA of human collagen is reversely transcribed to generate cDNA and then expressed in escherichia coli BL21, the structure and the obtaining method can refer to patent application files with the patent number ZL01106757.8 and the patent name of human-like collagen and the production method thereof, and the relative molecular mass of the recombinant collagen is 97kDa;
Step 102, dropwise adding 40mL of glycidyl methacrylate into the recombinant collagen solution, stirring and reacting for 8 hours in a 37 ℃ water bath, and collecting a product;
Step 103, continuously dialyzing the product in deionized water for 7 days, and freeze-drying to obtain the methacryloyl recombinant collagen, wherein the cutoff molecular weight of the dialysis bag is 3500Da;
step two, providing the methacryloylated hyaluronic acid, which specifically comprises the following steps:
Step 201, 1.5g of sodium hyaluronate is dissolved in 100mL of deionized water to obtain a hyaluronic acid solution, wherein the relative molecular mass of the sodium hyaluronate is 500kDa;
Step 202, dropwise adding 30mL of glycidyl methacrylate into the hyaluronic acid solution, stirring and reacting for 8 hours in a water bath at 60 ℃, and collecting a product;
Step 203, continuously dialyzing the product in deionized water for 7 days, and freeze-drying to obtain the methacryloyl hyaluronic acid, wherein the cutoff molecular weight of the dialysis bag is 3500Da;
step three, providing 3D printing biological ink for preparing artificial blood vessels, which specifically comprises the following steps:
Step 301, placing 250mg of methacryloyl recombinant collagen and 5mg of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP) in 2.5mL of deionized water, sealing with tinfoil to avoid light, and stirring at room temperature until the materials are completely dissolved to obtain solution A, wherein the stirring time at room temperature can be 12 hours;
step 302, placing 75mg of methacryloyl hyaluronic acid in 2.5mL of deionized water, and stirring in a 37 ℃ water bath until the hyaluronic acid is completely dissolved to obtain solution B, wherein the stirring time in the water bath can be 2 hours;
step 303, mixing the solution A in the step 301 and the solution B in the step 302, and continuing stirring for 1h to obtain a gel solution, wherein the gel solution has no obvious fluidity and is milky;
Step 304, standing the gel solution in a refrigerator at 4 ℃ for about 12 hours to obtain gel biological ink, and standing the gel solution at 4 ℃ for about 12 hours to fully crosslink and defoam the gel solution;
The method comprises the steps of taking gel bio-ink as a raw material to prepare a 3D printing bracket for preparing an artificial blood vessel, and specifically comprises the steps of filling the gel bio-ink into a charging barrel of a 3D printer, performing 3D printing to obtain the 3D printing bracket, wherein the model of the 3D printer is FOODBOT-S2, extruding the gel bio-ink, contacting ultraviolet light (365 nm,10mW/cm 2) after printing, irradiating for 5min for photo-crosslinking and curing, and irradiating and sterilizing Co60 to obtain the 3D printing hydrogel bracket for preparing the artificial blood vessel, namely a bracket C, wherein the diameter of the bracket C is 6mm and the height of the bracket C is 1cm;
The specification of the nozzle of the 3D printer is 23G;
The printing speed is 30mm/s;
The printing temperature is 25 ℃;
The extrusion amount was 8%.
The properties of the 3D printed hydrogel scaffold for preparing vascular prostheses of this example were substantially identical to those of example 1.
Example 4
This example is the same as example 1, except that in step four, the nozzle specification of the 3D printer is 23G, the printing speed is 20mm/s, the extrusion amount is 4%, and the crosslinking time is 1min.
Example 5
This example is the same as example 1, except that in step four, the nozzle specification of the 3D printer is 26G, the printing speed is 20mm/s, the extrusion amount is 8%, and the crosslinking time is 1min.
Examples 4 and 5 each gave a 3D printed hydrogel scaffold, and the hydrogel scaffold of example 1 had a more uniform and symmetrical scaffold structure from the viewpoint of the scaffold structure, indicating that the size, extrusion amount per unit time and printing speed of the bio-ink extruded filaments of the present invention affect the morphology of the 3D printed hydrogel scaffold.
Evaluation of Performance
Synthesis of physicochemical Properties of the Components
FIG. 1 is a schematic flow chart of the preparation of the methacryloylated recombinant collagen according to example 1 of the present invention, in which glycidyl methacrylate was added dropwise to a hyaluronic acid solution, and uniform transparent methacryloylated hyaluronic acid was formed under a water bath condition of 37 ℃. FIG. 2 is a schematic diagram showing the gel formation of the methacryloylated recombinant collagen prepared in example 1 of the present invention after ultraviolet irradiation under the action of a photoinitiator. As can be seen from fig. 2, the methacryloylated recombinant collagen according to the invention can be glued under light conditions. FIG. 3 is a chart showing the Fourier transform infrared absorption spectrum analysis of the methacryloylated recombinant collagen prepared in example 1 of the present invention. As can be seen from FIG. 3, there is a stretching vibration peak of the carbon-carbon double bond at 1653cm -1, indicating successful methacryloylation of the recombinant collagen. FIG. 4 shows the result of 1 H-NMR analysis of methacryloylated recombinant collagen prepared in example 1 of the present invention. As can be seen from FIG. 4, there is a bimodal olefin at 6.08ppm,5.70ppm, confirming that the recombinant collagen was successfully grafted with double bond groups.
FIG. 5 is a schematic diagram showing the procedure for preparing a methacryloylated hyaluronic acid according to example 1 of the present invention, in which glycidyl methacrylate was added dropwise to a hyaluronic acid solution, and a homogeneous transparent methacryloylated hyaluronic acid was formed under the condition of a water bath at 60 ℃. FIG. 6 is a schematic diagram showing the gel formation of the methacryloylated hyaluronic acid prepared in example 1 of the present invention after irradiation with ultraviolet light under the action of a photoinitiator. As can be seen from fig. 6, the methacryloylated hyaluronic acid of the invention can be glued under light conditions. Referring to fig. 2, the methacryloylated recombinant collagen and methacryloylated hyaluronic acid of the present invention can be used as the backbone structure of hydrogels. FIG. 7 is a chart showing the Fourier transform infrared absorption spectrum of the methacryloylated hyaluronic acid prepared in example 1 of the present invention. As can be seen from FIG. 7, there is a stretching vibration peak of the carbon-carbon double bond at 1710cm -1, indicating successful methacryloylation of hyaluronic acid. FIG. 8 shows the result of 1 H-NMR analysis of methacryloylated hyaluronic acid prepared in example 1 of the invention. As can be seen from FIG. 8, there is a bimodal olefin at 6.17ppm,5.74ppm, confirming the successful grafting of the double bond groups to the hyaluronic acid.
Physical and chemical properties of gel biological ink for preparing artificial blood vessels
The rheological properties of the scaffolds prepared in example 1 and example 2 were tested. The testing method comprises the steps of placing the gel biological ink in the step 303 of the embodiment 1 or the embodiment 2 on a rotary rheometer circular table, testing the extrudability of the gel by using steady-state speed scanning (0.1-1000 s -1), and testing the stability of the hydrogel by using oscillation frequency scanning (0.1-100 Hz and strain of 1%) of the bracket A in the embodiment 1 and the embodiment 2. Fig. 9 is a schematic diagram showing the results of the extrudability test of the gel bio-ink of example 1 and example 2, and it can be seen from fig. 9 that the viscosity of the samples showed a decreasing trend with increasing shear rate, indicating that the samples had shear thinning characteristics and printability.
Fig. 10 is a schematic diagram of the stability test results of the stents of example 1 and example 2. According to FIG. 10, the storage modulus (G ') and loss modulus (G') of the samples were substantially unchanged with the change in oscillation frequency, indicating that the scaffold had solid-like elasticity, confirming the stability of the hydrogel scaffold of the present invention.
FIG. 11 is a cross-sectional scanning electron microscope image of a100 μm sub-stent A according to example 1, and the test method comprises freeze-drying the hydrogel for preparing artificial blood vessels, cutting into samples with a thickness of about 1mm, adhering the samples to a sample stage with a conductive adhesive, spraying gold, and observing the microscopic morphology thereof by a Scanning Electron Microscope (SEM). As can be seen from fig. 11, the stent is printed layer by a 3D printer.
FIG. 12 is a surface scanning electron microscope image of a 100 μm sub-stent A according to example 1, and the test method comprises freeze-drying the hydrogel for preparing artificial blood vessels, cutting into samples with a thickness of about 1mm, adhering the samples to a sample stage with a conductive adhesive, spraying gold, and observing the microscopic morphology by a Scanning Electron Microscope (SEM). As can be seen from fig. 12, the scaffold a has a three-dimensional pore structure, which can provide a microenvironment for endothelial cell growth;
Fig. 13 is a topography diagram of a support a of example 1, and the testing method includes loading the bio-ink into a cartridge of a 3D printer, and performing 3D printing to obtain a 3D printing support. As can be seen from fig. 13, the stent a has a stent diameter of 6mm and a height of 5mm;
Cell compatibility
FIG. 14 is a schematic diagram showing the cytotoxicity test results of the scaffold A of example 1, and the test method thereof comprises:
Preparing DMEMF a complete culture solution, specifically comprising adding fetal bovine serum and diabody into DMEMF12 basic culture medium to obtain DMEMF a complete culture solution, wherein the weight percentage of the fetal bovine serum in DMEMF a complete culture solution is 10%, the weight percentage of the diabody is 1%, and the diabody is streptomycin and penicillin;
Preparing stent leaching solution, which specifically comprises placing stent A of example 1 in a 50mL sterile centrifuge tube, adding DMEMF complete culture solution into DMEMF mL of complete culture solution for each 0.1g of stent A, placing the centrifuge tube in an incubator with 37 ℃ and 5% CO 2, and culturing for 72h to obtain stent leaching solution;
HUVECs are inoculated into a 96-well plate at the density of 1X 10 4 cells/well, cultured for 24 hours in an incubator with 37 ℃ and 5% CO 2, old culture solution is sucked off, stent leaching solution is added for continuous culture, DMEM complete culture solution is used as a control group, CCK-8 solution is added into the 96-well plate according to 10 mu L/well after the culture for 24 hours and 48 hours respectively, after continuous culture for 2-4 hours, supernatant is sucked out, OD value of each well is measured at 450nm by an enzyme-labeled instrument, cell survival rate is calculated according to the OD value, and cell viability is calculated as follows:
The calculation result of the cell viability is shown in fig. 14, and it can be seen that the 3D scaffold material prepared by the present invention has no obvious cytotoxicity and can be used for organisms.
Cell migration promoting Property
FIGS. 15 and 16 are schematic diagrams showing the results of the cell migration promoting ability test of the hydrogel for preparing an artificial blood vessel of example 1. The test method comprises the steps of placing hydrogel and low-serum DMEMF culture solution into a 50mL sterile centrifuge tube to obtain a sample, wherein the hydrogel concentration in the sample is 0.1g/mL, the low-serum DMEMF culture solution contains DMEMF basic culture medium and 2% fetal bovine serum, placing the centrifuge tube with the sample into a 37 ℃ and 5% CO 2 incubator for 72 hours to obtain hydrogel leaching solution, inoculating HUVECs into a 6-hole plate at the density of 1.2X10 6 cells/hole, after the cells are fully distributed on the bottom of the 6-hole plate, using a 10 mu L pipette tip to vertically scratch, sucking out old culture solution, washing twice with PBS, adding the hydrogel leaching solution and the low-serum DMEMF culture solution respectively, obtaining an experimental group and a control group, and after culturing for 0h, 12h and 24h, randomly selecting three areas by using an inverted microscope for photographing and quantifying by Image J software. Cell mobility was calculated as follows:
The area of the scratch area is shown in fig. 15, the photographed result is shown in fig. 16, and it can be seen from fig. 15 and 16 that the mobility of the hydrogel group of the present invention is 64.21 ±2 (%), and the ability to promote migration of HUVECs is significantly improved.
Blood compatibility
Fig. 17 and 18 are blood compatibility test results of the hydrogel for preparing an artificial blood vessel of example 1. The test method includes centrifuging (5000 rpm,5 min) the erythrocytes and washing three times with physiological saline, followed by dilution to a 5% (v/v) solution. Physiological saline was added to the sterilized stent sample at a concentration of 0.1g/mL, and the mixture was immersed at 37℃for 24 hours to obtain a hydrogel extract, and then 800. Mu.L of the hydrogel extract and 200. Mu.L of the red blood cell suspension were mixed. Deionized water and physiological saline were added to the control group, respectively. After incubation at 37℃for 1 hour, centrifugation (2000 rpm,5 min) was performed and the absorbance of the supernatant was measured at 540nm using a microplate reader. The hemolysis rate was calculated as follows:
Wherein As, an and Ap represent absorbance values of hydrogel group, physiological saline (negative group), deionized water (positive group), respectively.
The photographing results are shown in fig. 17, the quantification results are shown in fig. 18, and it can be seen from fig. 17 and 18 that the hemolysis rate of the hydrogel group of the present invention is less than 0.2%, which demonstrates the biosafety of the prepared scaffold material.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent structural changes of the above embodiment according to the technical matter of the present invention still fall within the scope of the technical solution of the present invention.
Claims (10)
1. The preparation method of the gel bio-ink for the 3D printing of the small-diameter artificial blood vessel is characterized by comprising the following steps of:
Step one, dissolving methacryloyl recombinant collagen and a photoinitiator in deionized water under a light-shielding condition to obtain solution A;
Step two, mixing the solution A and the solution B in the step one to obtain gel solution;
and thirdly, standing the gel liquid in the second step in a refrigerator at the temperature of 4 ℃ for 8-36 hours to obtain the gel biological ink for 3D printing of the small-diameter artificial blood vessel.
2. The preparation method of the gel bio-ink for the 3D printing of the small-diameter artificial blood vessel, which is disclosed in claim 1, is characterized by specifically comprising the steps of dripping glycidyl methacrylate into a recombinant collagen solution, stirring and reacting for 6-8 hours in a 37 ℃ water bath, dialyzing, and freeze-drying to obtain the methacryloylated recombinant collagen.
3. The preparation method of the gel bio-ink for the 3D printing of the small-diameter artificial blood vessel is characterized in that the recombinant collagen solution is obtained by dissolving recombinant collagen in deionized water, the mass-volume concentration of the recombinant collagen solution is 1% -2%, the relative molecular mass of the recombinant collagen is 97kDa, and the dosage of the glycidyl methacrylate is 10-30 mL of glycidyl methacrylate for each g of the recombinant collagen.
4. The preparation method of the gel bio-ink for 3D printing of small-diameter artificial blood vessels, which is disclosed in claim 1, is characterized by specifically comprising the steps of dropwise adding glycidyl methacrylate into a hyaluronic acid solution, stirring and reacting for 6-8 hours in a water bath at 60 ℃, dialyzing, and freeze-drying to obtain the methacryloylated hyaluronic acid.
5. The preparation method of the gel bio-ink for the 3D printing of the small-diameter artificial blood vessel is characterized in that the hyaluronic acid solution is obtained by dissolving sodium hyaluronate in deionized water, the mass-volume concentration of the hyaluronic acid solution is 1% -2%, the relative molecular mass of the sodium hyaluronate is 10-500 kDa, and the dosage of the glycidyl methacrylate is 10-30 mL of the glycidyl methacrylate per g of the sodium hyaluronate.
6. The method for preparing gel bio-ink for 3D printing small-diameter vascular prosthesis according to claim 1, wherein the photoinitiator in the first step is phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate.
7. The method for preparing the gel bio-ink for 3D printing of small-diameter artificial blood vessels according to claim 1, wherein in the second step, the mass-volume concentration of the methacryloyl recombinant collagen in the gel solution is 5% -10%, the mass-volume concentration of the methacryloyl hyaluronic acid is 1.5% -2%, and the mass-volume concentration of the photoinitiator is 0.1% -0.25%.
8. Use of a gel bio-ink prepared according to the method of claim 1 for preparing a 3D printing scaffold for preparing an artificial blood vessel.
9. The application of the 3D printing bracket for preparing the artificial blood vessel, which is characterized in that the preparation method for the 3D printing bracket for preparing the artificial blood vessel comprises the steps of filling the gel bio-ink into a charging barrel of a 3D printer for 3D printing, contacting ultraviolet light after the gel bio-ink is extruded and irradiating for 3-5 min for photo-crosslinking and curing, and sterilizing to obtain the 3D printing bracket for the artificial blood vessel.
10. The use according to claim 9, wherein the 3D printer has a nozzle specification of 23g to 26g, a printing speed of 10mm/s to 30mm/s, a printing temperature of 25 ℃, and an extrusion amount of 4% to 8%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411444696.7A CN119318736A (en) | 2024-10-16 | 2024-10-16 | Preparation method and application of gel biological ink for 3D printing of small-diameter artificial blood vessels |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411444696.7A CN119318736A (en) | 2024-10-16 | 2024-10-16 | Preparation method and application of gel biological ink for 3D printing of small-diameter artificial blood vessels |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN119318736A true CN119318736A (en) | 2025-01-17 |
Family
ID=94233995
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411444696.7A Pending CN119318736A (en) | 2024-10-16 | 2024-10-16 | Preparation method and application of gel biological ink for 3D printing of small-diameter artificial blood vessels |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN119318736A (en) |
-
2024
- 2024-10-16 CN CN202411444696.7A patent/CN119318736A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113679888B (en) | Photo-curing molding composite hydrogel matrix precursor, preparation method thereof and stent with same | |
| CN108653809B (en) | A composite hydrogel based on black phosphorus and gelatin and its application in bone tissue engineering | |
| Gatenholm et al. | Bacterial nanocellulose as a renewable material for biomedical applications | |
| Rezaei et al. | 3D printed chitosan/polycaprolactone scaffold for lung tissue engineering: hope to be useful for COVID-19 studies | |
| CN110790950A (en) | Photo-crosslinking recombinant collagen hydrogel, preparation method and application thereof in 3D bioprinting | |
| Wang et al. | Improvement in physical and biological properties of chitosan/soy protein films by surface grafted heparin | |
| CN112999425B (en) | Double-layer hydrogel tubular tissue engineering scaffold and preparation method thereof | |
| Li et al. | Injectable thermosensitive chitosan/gelatin-based hydrogel carried erythropoietin to effectively enhance maxillary sinus floor augmentation in vivo | |
| CN114225096A (en) | A composite hydrogel for promoting wound healing and its preparation method and application | |
| Choi et al. | Evaluation of hyaluronic acid/agarose hydrogel for cartilage tissue engineering biomaterial | |
| CN106674486B (en) | A kind of polyester type polyurethane material of side chain group containing Phosphorylcholine and preparation method thereof | |
| Davis et al. | Modified PHEMA hydrogels | |
| Zhang et al. | Advances in photocrosslinkable materials for 3D bioprinting | |
| Yan et al. | Ethanol-lubricated expanded-polytetrafluoroethylene vascular grafts loaded with eggshell membrane extract and heparin for rapid endothelialization and anticoagulation | |
| Arisoly Xavier Acasigua et al. | Novel chemically modified bacterial cellulose nanocomposite as potential biomaterial for stem cell therapy applications | |
| CN109106987B (en) | A kind of bone implant with both mechanical adaptability and osteogenic activity and preparation method thereof | |
| Yang et al. | Double-modified bacterial cellulose/soy protein isolate composites by laser hole forming and selective oxidation used for urethral repair | |
| Hu et al. | Production of novel elastic bacterial nanocellulose/polyvinyl alcohol conduits via mercerization and phase separation for small-caliber vascular grafts application | |
| Zineh et al. | Experimental and numerical study on the performance of printed alginate/hyaluronic acid/halloysite nanotube/polyvinylidene fluoride bio-scaffolds | |
| CN108310463B (en) | A kind of 3D printing biological ink and preparation method thereof | |
| Chang et al. | 3D bioprinted pectin-based hydrogel as sustainable biomaterials for musculoskeletal tissue engineering | |
| Liu et al. | Hydrophilic silsesquioxane nanocages toughened extracellular matrix biomimetic Poly (γ-Glutamic acid) multidimensional self-polymerizable and osteogenic hybrid hydrogel for osteoporotic bone regeneration | |
| CN115105631A (en) | Photopolymerization artificial exosome blood vessel prepared by cold casting method, and preparation method and application thereof | |
| CN108452378B (en) | 3D biological printing forming method | |
| CN108084466B (en) | A composite membrane based on egg white and methacrylic acid derivatized polymer and its application in culturing stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |