CN113350577A - 3D printing composite hydrogel support, preparation method thereof and sterile freeze-drying support - Google Patents
3D printing composite hydrogel support, preparation method thereof and sterile freeze-drying support Download PDFInfo
- Publication number
- CN113350577A CN113350577A CN202110661457.7A CN202110661457A CN113350577A CN 113350577 A CN113350577 A CN 113350577A CN 202110661457 A CN202110661457 A CN 202110661457A CN 113350577 A CN113350577 A CN 113350577A
- Authority
- CN
- China
- Prior art keywords
- printing
- composite hydrogel
- scaffold
- solution
- support
- 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
- 239000000017 hydrogel Substances 0.000 title claims abstract description 56
- 239000002131 composite material Substances 0.000 title claims abstract description 45
- 238000010146 3D printing Methods 0.000 title claims abstract description 22
- 238000004108 freeze drying Methods 0.000 title claims description 14
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 108010010803 Gelatin Proteins 0.000 claims abstract description 30
- 239000008273 gelatin Substances 0.000 claims abstract description 30
- 229920000159 gelatin Polymers 0.000 claims abstract description 30
- 235000019322 gelatine Nutrition 0.000 claims abstract description 30
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 30
- DZGWFCGJZKJUFP-UHFFFAOYSA-N Tyramine Natural products NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229960003732 tyramine Drugs 0.000 claims abstract description 17
- 239000010949 copper Substances 0.000 claims abstract description 16
- 239000002105 nanoparticle Substances 0.000 claims abstract description 15
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 14
- 108010022355 Fibroins Proteins 0.000 claims abstract description 14
- 229910052802 copper Inorganic materials 0.000 claims abstract description 14
- 230000001737 promoting effect Effects 0.000 claims abstract description 9
- 238000007639 printing Methods 0.000 claims description 45
- 239000000243 solution Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 239000011259 mixed solution Substances 0.000 claims description 19
- 239000008367 deionised water Substances 0.000 claims description 15
- 229910021641 deionized water Inorganic materials 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 12
- 238000002791 soaking Methods 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 11
- 230000010478 bone regeneration Effects 0.000 claims description 7
- PRQROPMIIGLWRP-BZSNNMDCSA-N chemotactic peptide Chemical compound CSCC[C@H](NC=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PRQROPMIIGLWRP-BZSNNMDCSA-N 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 230000005855 radiation Effects 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- 239000007987 MES buffer Substances 0.000 claims description 2
- 230000005251 gamma ray Effects 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 claims 1
- 210000000988 bone and bone Anatomy 0.000 abstract description 13
- 238000006731 degradation reaction Methods 0.000 abstract description 10
- 230000015556 catabolic process Effects 0.000 abstract description 9
- 230000017423 tissue regeneration Effects 0.000 abstract description 6
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 abstract description 5
- 229910001431 copper ion Inorganic materials 0.000 abstract description 5
- 230000033115 angiogenesis Effects 0.000 abstract description 4
- 230000006835 compression Effects 0.000 abstract description 4
- 238000007906 compression Methods 0.000 abstract description 4
- 238000011065 in-situ storage Methods 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- DZGWFCGJZKJUFP-UHFFFAOYSA-O tyraminium Chemical compound [NH3+]CCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-O 0.000 abstract 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 5
- 230000007547 defect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000835 fiber Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000009835 boiling Methods 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- UJMYYOYPZCSYDQ-UHFFFAOYSA-N ethanesulfonic acid morpholine hydrate Chemical compound O.C(C)S(=O)(=O)O.N1CCOCC1 UJMYYOYPZCSYDQ-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 239000005543 nano-size silicon particle Substances 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000011960 computer-aided design Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 239000010954 inorganic particle Substances 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 102100032742 Histone-lysine N-methyltransferase SETD2 Human genes 0.000 description 1
- 101000654725 Homo sapiens Histone-lysine N-methyltransferase SETD2 Proteins 0.000 description 1
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 1
- 101001055320 Myxine glutinosa Insulin-like growth factor Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000005313 bioactive glass Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 230000001808 coupling effect Effects 0.000 description 1
- 230000009193 crawling Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000013081 microcrystal Substances 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 230000009818 osteogenic differentiation Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- -1 silicon ions Chemical class 0.000 description 1
- 239000012890 simulated body fluid Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000012876 topography Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000007998 vessel formation Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H1/00—Macromolecular products derived from proteins
-
- 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
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
-
- 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
-
- 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
- A61L27/58—Materials at least partially resorbable by the body
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
- A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
- A61L2300/624—Nanocapsules
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Animal Behavior & Ethology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Dispersion Chemistry (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Composite Materials (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a 3D printing composite hydrogel scaffold, a preparation method thereof and an aseptic freeze-dried scaffold, and particularly relates to a preparation method and application of a scaffold for promoting vascularized bone tissue regeneration, which is prepared by performing 3D printing on silk fibroin, tyramine modified gelatin and element-doped copper inorganic micro-nano particles as raw materials. The composite hydrogel support comprises 0.1-10 wt% of element-doped copper inorganic micro-nano particles, 10-20 wt% of tyramine modified gelatin (Tyr-Gel) and 5-7 wt% of regenerated silk fibroin. According to the invention, the fibroin is compounded to endow the scaffold with good compression strength and degradation performance, and the doping of the element-doped copper inorganic micro-nano particles endows the scaffold with the activity of promoting angiogenesis and bone tissue regeneration, so that the problems that the element-doped copper inorganic micro-nano particles are difficult to directly perform 3D printing and realize in-situ micro copper ion release are solved.
Description
Technical Field
The invention relates to the technical field of biomedical 3D printing tissue engineering scaffolds, in particular to a 3D printing composite hydrogel scaffold capable of promoting vascularized bone regeneration, a preparation method thereof and a sterile freeze-drying scaffold.
Background
The tissue engineering bone prepared by using the 3D printing technology has high designability, can be used for preparing a tissue engineering scaffold which is highly matched with bone defects of a patient by combining a medical image programming technology, a CAD (computer-aided design) technology and a 3D printing technology, and the porosity, the mechanical strength and the biological performance of the scaffold can be realized by selecting and compounding materials and printing the structural design of the scaffold, and can also avoid the defects of donor parts, the morbidity of the donor parts and the immune rejection caused by autologous bone transplantation and allogeneic bone transplantation, so that the tissue engineering bone has wide application potential in the field of bone tissue regeneration. The porosity, pore size and wall thickness of the stent are known to have a significant effect on vascular and tissue ingrowth.
The silicon-based nanoparticles have high specific surface area and abundant surface silicon hydroxyl groups, when a proper amount of the silicon-based nanoparticles are compounded into the hydrogel of the continuous phase, the friction force between an inorganic phase and an organic continuous phase is increased due to the increase of the specific surface area and the formation of hydrogen bonds, the viscosity of the system is increased, and therefore the printability and the fidelity can be improved. Meanwhile, the mechanical strength of the material can be effectively improved by doping the inorganic particles, so that the material can better meet the mechanical requirements of bone implants. The silicon ions released by the silicon-based nanoparticles in the slow degradation process have the function of stimulating intercellular communication, can promote the expression of angiogenesis-related growth factors such as VEGF, bFGF and IGF, and promote the budding, development, fusion and maturation of capillary networks.
Copper ions can simulate a hypoxia environment to activate an HIF-1 pathway to promote the expression of angiogenesis-related genes and promote angiogenesis and tissue regeneration and repair. The trace copper ions with proper concentration can directly promote the osteogenic differentiation of the bone marrow mesenchymal stem cells. Therefore, the inorganic particles doped with copper elements, such as Cu-BG, Cu-BG or natural copper-containing inorganic micro-nano particles, are compounded into the hydrogel, so that biocompatibility and bioactivity can be considered, in-situ release of copper ions is realized, physiological toxicity caused by overhigh serum copper level is avoided, and the coupling effect of antibiosis and blood vessel formation and bone formation can be realized.
The hydrogel is a hydrophilic gelatinous polymer material with high water content and a three-dimensional network cross-linked structure, and has the characteristics of being capable of rapidly swelling to a balanced volume in an aqueous solution without being dissolved and still keeping the shape and the space structure of the hydrogel. Gelatin is a widely used natural polymer, the molecular chain of the gelatin has a cell recognition site RGD sequence, the gelatin can promote the adhesion, extension and crawling of cells, the gelatin has good biocompatibility, and the molecular structure of the gelatin is designed to endow the material with proper degradation rate, water absorption, mechanical properties and the like such as methacrylic acid gelatin (GelMA). The silk fibroin is a tough and elastic protein, and crystalline regions and amorphous regions exist in macromolecules of the silk fibroin, so that the silk fibroin can form nano-microcrystals through beta-sheet conformation transformation, and the overall mechanical strength is improved.
In conclusion, aiming at the problems of the existing bone defect repair scaffold, the 3D printing composite hydrogel scaffold which is more in line with the requirements of an ideal bone tissue engineering scaffold, has strong designability, advanced manufacturing mode and simple raw material acquisition and can promote the regeneration of vascularized bone tissues is very important.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a 3D printing composite hydrogel scaffold capable of promoting vascularized bone regeneration, which has the advantages of easily obtained raw materials, high mechanical strength, good biocompatibility, high bioactivity and the like.
The second purpose of the invention is to provide a preparation method of the 3D printing composite hydrogel scaffold capable of promoting vascularized bone regeneration.
The third purpose of the invention is to provide a sterile freeze-drying scaffold prepared by using the composite hydrogel scaffold, which can be used for animal experiments.
The first purpose of the invention is realized by the following technical scheme: A3D printing composite hydrogel scaffold capable of promoting vascularized bone regeneration comprises 0.1-10 wt% of element-doped copper inorganic micro-nano particles, 10-20 wt% of tyramine modified gelatin (Tyr-Gel) and 5-7 wt% of regenerated silk fibroin.
Further, the element-doped copper inorganic micro-nano particles are Cu-BG or Cu-MSN, and the mass fraction of the Cu element in the inorganic micro-nano particles is 0.1-10%.
The second purpose of the invention is realized by the following technical scheme: a preparation method of a 3D printing composite hydrogel scaffold capable of promoting vascularized bone regeneration comprises the following steps:
1) dissolving gelatin in MES buffer solution, adding tyramine hydrochloride and EDC/NHS, reacting at normal temperature to obtain tyramine modified gelatin solution, freeze-drying to obtain tyramine modified gelatin, and dissolving the tyramine modified gelatin in regenerated silk fibroin to obtain mixed solution A;
2) dispersing element-doped copper inorganic micro-nano particles in a deionized water solution, performing ultrasonic treatment by using an ultrasonic machine, and then homogenizing at a high speed by using a homogenizer to obtain a uniformly dispersed mixed solution B;
3) gradually dripping the mixed solution B into the mixed solution A at a specific stirring speed to obtain a mixed solution C;
4) adding a high-concentration HRP aqueous solution into the mixed solution C at a specific stirring speed, and uniformly stirring to obtain a mixed solution D, namely printing ink;
5) the mixed solution D is stored at a constant temperature for 3-12 hours, so that the printing ink has proper viscosity to support printing;
6) and transferring the printing ink which is stored stably at the constant temperature into a printing material cylinder, printing according to a set program, and receiving the platform temperature of 4-20 ℃ to obtain the printed composite hydrogel support.
The third purpose of the invention is realized by the following technical scheme: a sterile freeze-dried scaffold prepared from composite hydrogel scaffold is prepared through immersing the printed composite hydrogel scaffold in H2O2Soaking in the solution for 30 min-2 h, then soaking the composite hydrogel support in an alcohol solution for processing overnight, and finally, freeze-drying the processed composite hydrogel support and sterilizing the composite hydrogel support by using gamma ray radiation to obtain the sterile freeze-dried support.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the material used in the invention is natural renewable polymer of gelatin and fibroin, the source of the material is wide and easy to obtain, and a more similar environment is provided for the growth and metabolism of the high water content cells of the hydrogel.
2. The preparation process is simple and easy to operate, and the preparation conditions are mild.
3. According to the invention, micro-nano element doped copper inorganic nanoparticles are uniformly dispersed in the hydrogel matrix, so that the agglomeration of micro-nano particles is overcome, and the printing property of ink and the mechanical property of a support are improved.
4. The composite hydrogel scaffold prepared by the invention can realize the release of in-situ copper ions, and effectively promote the regeneration of blood vessels and the regeneration of bone tissues.
5. The composite hydrogel scaffold prepared by the invention has good mechanical property and degradation property, and can play a supporting role in the tissue regeneration period.
Drawings
FIG. 1 is a motor microscope image of different Cu-BG composite hydrogel scaffolds.
FIG. 2 is a scanning electron microscope image of different Cu-BG composite hydrogel scaffolds.
FIG. 3 is a graph of the compression curves of different Cu-BG composite hydrogel scaffolds.
FIG. 4 is a graph showing degradation curves of different Cu-BG composite hydrogel scaffolds.
FIG. 5 is a laser confocal picture of endothelial cells cultured by different Cu-BG composite hydrogel scaffolds.
Detailed Description
The present invention is described in further detail below with reference to a number of examples, but the embodiments of the present invention are not limited thereto.
Example 1
Dissolving 5.4g of morpholine ethanesulfonic acid monohydrate in 500ml of deionized water, stirring for dissolving, heating to 50 ℃, adding 10g of pigskin-derived gelatin, cooling to room temperature, adding 5g of tyramine hydrochloride, adding 0.74g/0.22g (EDC/NHS) to activate carboxyl, reacting for 12h to obtain tyramine-modified gelatin, dialyzing for 4d, and freeze-drying for later use. The chemical structure of the tyramine modified gelatin dissolved in deionized water is analyzed by using nuclear magnetic hydrogen spectroscopy, and the characteristic peak belonging to tyramine groups relative to the gelatin without modification is observed to appear on the nuclear magnetic hydrogen spectrum curve of the tyramine modified gelatin to indicate the success of modification of tyramine groups.
Dissolving degummed silk in 9.3M lithium bromide solution, boiling for 4h at 60 ℃, then transferring into a dialysis bag of 8000-14000, dialyzing for 1d with deionized water, changing water for 4 times, and properly diluting to obtain 6% fibroin aqueous solution.
Dissolving 0.005g of Cu-BG in 5ml of deionized water, carrying out ultrasonic treatment for 30min multiplied by 3 times by using an ultrasonic instrument, and then carrying out high-speed homogenization for 5min by using a high-speed homogenizer at 10krpm to obtain a uniform Cu-BG aqueous solution.
0.5g of Tyr-Gel was dissolved in 10ml of a 6% SF solution, heated at 60 ℃ to dissolve it, and then the uniform Cu-BG solution was added dropwise, and 100. mu.L of a 3000U/L HRP aqueous solution was added dropwise depending on the volume of the resulting mixed solution. Stirring at medium speed to make it uniform, and keeping the temperature in a water bath kettle at 29 ℃ for 3h to be used for 3D printing.
The printing ink is transferred into a printing material cylinder, the heat preservation temperature of the printing head is 29 ℃, the printing pressure is 0.8bar, the printing speed is 12mm/s, the inner diameter of the printing needle is 260 mu m, the printing model is a cylindrical support with the diameter of R10mmH3, the fiber spacing is 500 mu m, and the temperature of a receiving platform is 4 ℃.
After printing was complete, the scaffolds were transferred to 5mM H at 5 ℃ ice together with the printing pad2O2Soaking in the solution for 30min, then carefully removing the stent from the pad, transferring to 75% methanol solution for treatment overnight, soaking the resulting stent in deionized water to replace methanol, freeze-drying the stent and sterilizing by gamma irradiation.
Example 2
Dissolving 5.4g of morpholine ethanesulfonic acid monohydrate in 500ml of deionized water, stirring for dissolving, heating to 50 ℃, adding 10g of pigskin-derived gelatin, cooling to room temperature, adding 5g of tyramine hydrochloride, adding 0.74g/0.22g (EDC/NHS) to activate carboxyl, reacting for 24h to obtain tyramine-modified gelatin, dialyzing for 4d, and freeze-drying for later use.
Dissolving degummed silk in 9.3M lithium bromide solution, boiling for 4h at 60 ℃, then transferring into a dialysis bag of 8000-14000, dialyzing for 1d with deionized water, changing water for 4 times, and properly diluting to obtain 5% fibroin aqueous solution.
Dissolving 0.1g of Cu-MSN in 5ml of deionized water, performing ultrasonic treatment for 30min multiplied by 3 times by using an ultrasonic instrument, and homogenizing by using a high-speed homogenizer to disperse to obtain a uniform Cu-MSN aqueous solution.
1.5g Tyr-Gel was dissolved in 10ml of 6% SF solution, heated at 60 ℃ to dissolve it, and then the above uniform Cu-MSN solution was added dropwise, and 100. mu.L of 3000U/L HRP aqueous solution was added dropwise depending on the volume of the resulting mixed solution. Stirring at medium speed to make it uniform, and keeping the temperature in a water bath kettle at 29 ℃ for 3h to be used for 3D printing.
The printing ink is transferred into a printing material cylinder, the heat preservation temperature of the printing head is 29 ℃, the printing pressure is 0.8bar, the printing speed is 12mm/s, the inner diameter of the printing needle is 260 mu m, the printing model is a cylindrical support with the diameter of R10mmH3, the fiber spacing is 500 mu m, and the temperature of a receiving platform is 4 ℃.
After printing was complete, the scaffolds were transferred to 5mM H at 5 ℃ ice together with the printing pad2O2Soaking in the solution for 30min, then carefully removing the stent from the pad, transferring to 75% methanol solution for treatment overnight, soaking the resulting stent in deionized water to replace methanol, freeze-drying the stent and sterilizing by gamma irradiation.
Example 3
Dissolving 5.4g of morpholine ethanesulfonic acid monohydrate in 500ml of deionized water, stirring for dissolving, heating to 50 ℃, adding 10g of pigskin-derived gelatin, cooling to room temperature, adding 5g of tyramine hydrochloride, adding 0.74g/0.22g (EDC/NHS) to activate carboxyl, reacting for 12h to obtain tyramine-modified gelatin, dialyzing for 4d, and freeze-drying for later use.
Dissolving degummed silk in 9.3M lithium bromide solution, boiling for 8h at 60 ℃, transferring into a dialysis bag of 8000-14000, dialyzing for 1d with deionized water, and changing water for 4 times to obtain 7% fibroin aqueous solution.
Dissolving 0.5g of Cu-BG in 5ml of deionized water, carrying out ultrasonic treatment for 30min multiplied by 3 times by using an ultrasonic instrument, and then carrying out high-speed homogenization for 5min by using a high-speed homogenizer at 10krpm to obtain a uniform Cu-BG aqueous solution.
3g of Tyr-Gel was dissolved in 14ml of a 7% SF solution, and the solution was heated at 60 ℃ to dissolve it, and then the uniform Cu-BG solution was added dropwise, and 400. mu.L of a 3000U/L HRP aqueous solution was added dropwise depending on the volume of the resulting mixed solution. Stirring at medium speed to make it uniform, and keeping the temperature in a water bath kettle at 29 ℃ for 3h to be used for 3D printing.
The printing ink is transferred into a printing material cylinder, the heat preservation temperature of the printing head is 29 ℃, the printing pressure is 2.0bar, the printing speed is 25mm/s, the inner diameter of the printing needle is 260 mu m, the printing model is a cylindrical support with the diameter of R10mmH3, the fiber spacing is 500 mu m, and the temperature of a receiving platform is 4 ℃.
After printing was complete, the scaffolds were transferred to 5mM H at 5 ℃ along with the printing pad2O2Soaking in the solution for 30min, then carefully removing the stent from the pad, transferring to 75% methanol solution for treatment overnight, soaking the resulting stent in deionized water to replace methanol, freeze-drying the stent and sterilizing by gamma irradiation.
Example 4 (micro-topography characterization of different Cu-BG doped composite hydrogel scaffolds)
Different Cu-BG are used for preparing printing ink according to the mass fraction of 1%, so that the 3D printing composite hydrogel scaffold is obtained and is completely swelled. Freezing at-20 deg.C, lyophilizing hydrogel with lyophilizer, and observing the pore structure of the scaffold with a microscope, as shown in FIG. 1. Cutting the section by using a scalpel blade, fixing the section on an electric microscope table by using conductive adhesive, spraying gold for 60s, and observing the section of each group of hydrogel support by using a scanning electron microscope so as to observe the internal appearance and the aperture rule of the hydrogel support. SEM photographs of the 3D-printed composite hydrogel scaffolds are shown in fig. 2. The results show that the composite hydrogel scaffold fibers are about 200 μm thick and have a pore size of about 500 μm.
Example 5 (mechanical testing of different Cu-BG doped composite hydrogel scaffolds)
Different Cu-BG are used for preparing printing ink according to the mass fraction of 1%, so that the 3D printing composite hydrogel scaffold is obtained and is completely swelled. A universal tester is used for carrying out compression performance test on the Cu-BG doped composite hydrogel after the different Cu-BG doped composite hydrogels are completely swelled, and the obtained compression curve is shown in figure 3. The result shows that the mechanical property of the hydrogel scaffold doped with SF is obviously improved, and the compressive strength and the modulus are obviously improved.
Example 6 (degradation testing of different Cu-BG doped composite hydrogel scaffolds)
Preparing printing ink by using different Cu-BG with the mass fraction of 1% to obtain a 3D printing composite hydrogel support, soaking the support in 1mg/ml of simulated body fluid according to the conversion of the mass of bioactive glass in the support, placing the support in an oven with the rotation speed of 120rpm and the temperature of 37 ℃ to perform a degradation experiment, changing the fluid every other day, and continuously performing the degradation experiment for 21 days, wherein the obtained degradation curve is shown in FIG. 4. The results show that the degradation rate of the group compounded with pure gelatin is significantly slowed down compared to the pure gelatin stent, and the mass loss rate at 21 days is about 30%.
Example 6 (cell compatibility test for different Cu-BG-doped composite hydrogel scaffolds)
Placing the lyophilized and radiation sterilized scaffold into 48-well plate, rehydrating with basic culture medium twice for 30min, and planting 2 × 10 on each well of scaffold5Human umbilical vein endothelial cells the hydrogel was injected into 48-well plates, placed in an incubator at 37 ℃ for 2h, the complete medium was added, and the solution was changed every two days.
The scaffolds cultured for 1,3, and 7 days were observed for viable and dead staining using a confocal laser microscope, and the confocal image is shown in FIG. 5. The results show that the number of endothelial cell adhesion proliferation on the 2Cu-BG scaffold is the largest and the best biocompatibility is shown after different days of culture of each group of composite hydrogel scaffolds.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (4)
1. The 3D printing composite hydrogel scaffold capable of promoting vascularized bone regeneration is characterized by comprising 0.1-10 wt% of element-doped copper inorganic micro-nano particles, 10-20 wt% of tyramine modified gelatin and 5-7 wt% of regenerated silk fibroin.
2. The 3D printing composite hydrogel scaffold capable of promoting vascularized bone regeneration according to claim 1, wherein the element-doped copper inorganic micro-nano particles are Cu-BG or Cu-MSN, and the mass fraction of Cu element in the inorganic micro-nano particles is 0.1-10%.
3. The method for preparing the 3D printing composite hydrogel scaffold as claimed in claim 1 or 2, which is characterized by comprising the following steps:
1) dissolving gelatin in MES buffer solution, adding tyramine hydrochloride and EDC/NHS, reacting at normal temperature to obtain tyramine modified gelatin solution, freeze-drying to obtain tyramine modified gelatin, and dissolving the tyramine modified gelatin in regenerated silk fibroin to obtain mixed solution A;
2) dispersing element-doped copper inorganic micro-nano particles in a deionized water solution, performing ultrasonic treatment by using an ultrasonic machine, and then homogenizing at a high speed by using a homogenizer to obtain a uniformly dispersed mixed solution B;
3) gradually dripping the mixed solution B into the mixed solution A at a specific stirring speed to obtain a mixed solution C;
4) adding a high-concentration HRP aqueous solution into the mixed solution C at a specific stirring speed, and uniformly stirring to obtain a mixed solution D, namely printing ink;
5) the mixed solution D is stored at a constant temperature for 3-12 hours, so that the printing ink has proper viscosity to support printing;
6) and transferring the printing ink which is stored stably at the constant temperature into a printing material cylinder, printing according to a set program, and receiving the platform temperature of 4-20 ℃ to obtain the printed composite hydrogel support.
4. A sterile lyophilized scaffold prepared using the composite hydrogel scaffold of claim 3, wherein:firstly, soaking the printed composite hydrogel scaffold in H2O2Soaking in the solution for 30 min-2 h, then soaking the composite hydrogel support in an alcohol solution for processing overnight, and finally, freeze-drying the processed composite hydrogel support and sterilizing the composite hydrogel support by using gamma ray radiation to obtain the sterile freeze-dried support.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110661457.7A CN113350577A (en) | 2021-06-15 | 2021-06-15 | 3D printing composite hydrogel support, preparation method thereof and sterile freeze-drying support |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110661457.7A CN113350577A (en) | 2021-06-15 | 2021-06-15 | 3D printing composite hydrogel support, preparation method thereof and sterile freeze-drying support |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113350577A true CN113350577A (en) | 2021-09-07 |
Family
ID=77534241
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110661457.7A Pending CN113350577A (en) | 2021-06-15 | 2021-06-15 | 3D printing composite hydrogel support, preparation method thereof and sterile freeze-drying support |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113350577A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116173308A (en) * | 2023-02-27 | 2023-05-30 | 苏州大学 | Injectable silk protein hydrogel capable of controlling release of metal ions and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150307728A1 (en) * | 2012-11-27 | 2015-10-29 | Tufts University | Biopolymer-based inks and use thereof |
| CN106806943A (en) * | 2016-03-31 | 2017-06-09 | 中国科学院上海硅酸盐研究所 | Formed in situ Injectable bio-active composite aquogel and its preparation method and application |
| CN109251323A (en) * | 2018-07-25 | 2019-01-22 | 华南理工大学 | Double cross-linked hydrogels of a kind of fibroin albumen-gelatin and preparation method thereof |
| CN110201225A (en) * | 2019-05-06 | 2019-09-06 | 华南理工大学 | 3D printing fibroin/gelatin bracket and preparation method thereof for repair of cartilage |
-
2021
- 2021-06-15 CN CN202110661457.7A patent/CN113350577A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150307728A1 (en) * | 2012-11-27 | 2015-10-29 | Tufts University | Biopolymer-based inks and use thereof |
| CN106806943A (en) * | 2016-03-31 | 2017-06-09 | 中国科学院上海硅酸盐研究所 | Formed in situ Injectable bio-active composite aquogel and its preparation method and application |
| CN109251323A (en) * | 2018-07-25 | 2019-01-22 | 华南理工大学 | Double cross-linked hydrogels of a kind of fibroin albumen-gelatin and preparation method thereof |
| CN110201225A (en) * | 2019-05-06 | 2019-09-06 | 华南理工大学 | 3D printing fibroin/gelatin bracket and preparation method thereof for repair of cartilage |
Non-Patent Citations (1)
| Title |
|---|
| ALESSANDRA BARI: ""Copper-containing mesoporous bioactive glass nanoparticles as multifunctional agent for bone regeneration"", 《ACTA BIOMATERIALIA》, 12 April 2017 (2017-04-12), pages 493 - 504, XP085034292, DOI: 10.1016/j.actbio.2017.04.012 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116173308A (en) * | 2023-02-27 | 2023-05-30 | 苏州大学 | Injectable silk protein hydrogel capable of controlling release of metal ions and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1698358A1 (en) | Transplantable biomaterial and method of preparing the same | |
| CN110790950A (en) | Photo-crosslinking recombinant collagen hydrogel, preparation method and application thereof in 3D bioprinting | |
| JP2022078243A (en) | Biogum and botanical gum hydrogel bioinks for physiological 3d bioprinting of tissue constructs for in vitro culture and transplantation | |
| CN103877617B (en) | Two cross-linked hydrogel of injectable fibroin protein-alginate and preparation method thereof and using method | |
| CN107007883B (en) | Cartilage repair support and preparation method thereof | |
| CN106075598A (en) | A kind of photo-crosslinking sericin hydrogel and its preparation method and application | |
| Jiang et al. | Preparation of cellulose nanofiber-reinforced gelatin hydrogel and optimization for 3d printing applications. | |
| EP1935438A1 (en) | Biomaterial for regenerative medicine | |
| CN112980001B (en) | Collagen composite hyaluronic acid gel, extracellular matrix bionic material and preparation method | |
| CN112972760B (en) | Endothelial extracellular matrix loaded 3D printing bone defect repair support and preparation method thereof | |
| CN105664260A (en) | Method for preparing bone tissue engineering three-dimensional porous support based on graphene/silk fibroin | |
| Sahranavard et al. | Three-dimensional bio-printing of decellularized extracellular matrix-based bio-inks for cartilage regeneration: a systematic review | |
| CN111228574A (en) | Tissue engineering meniscus scaffold and preparation method thereof | |
| CN111166937A (en) | Acellular extracellular matrix, preparation method thereof and biological ink | |
| CN106492281B (en) | Biocompatible bone graft and preparation method thereof | |
| CN113082295B (en) | Derived scaffold based on skin-derived acellular matrix and construction method thereof | |
| CN108992709B (en) | Acellular nerve matrix material and preparation method and application thereof | |
| CN109106987B (en) | Bone implant with mechanical adaptability and osteogenic activity and preparation method thereof | |
| CN102813961A (en) | Injection gel containing submicron hyaluronic acid microspheres and preparation method thereof | |
| JP6240997B2 (en) | Method for producing hydrogel film after vitrification, method for producing hydrogel material, hydrogel film after vitrification, dried hydrogel film after vitrification, and cell sheet | |
| CN113350577A (en) | 3D printing composite hydrogel support, preparation method thereof and sterile freeze-drying support | |
| Wang et al. | Elastic fiber-Reinforced silk fibroin scaffold with a Double-Crosslinking network for human ear-shaped cartilage regeneration | |
| CN108452378B (en) | 3D biological printing forming method | |
| CN111282021B (en) | Meniscus composite scaffold and preparation method thereof | |
| CN113679884A (en) | Tissue engineering hydrogel scaffold for promoting cell migration and preparation method thereof, and 3D printing slurry and preparation method thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210907 |
|
| RJ01 | Rejection of invention patent application after publication |