CN112618792B - Three-dimensional stent with communicated hollow structure and preparation method thereof - Google Patents
Three-dimensional stent with communicated hollow structure and preparation method thereof Download PDFInfo
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- CN112618792B CN112618792B CN202011486923.4A CN202011486923A CN112618792B CN 112618792 B CN112618792 B CN 112618792B CN 202011486923 A CN202011486923 A CN 202011486923A CN 112618792 B CN112618792 B CN 112618792B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- 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
Abstract
The invention discloses a three-dimensional stent and a preparation method thereof, wherein the preparation method comprises the following steps: compounding cells and alginate hydrogel to prepare 3D printing biological ink; constructing an active scaffold with a three-dimensional structure through 3D printing; carrying out multivalent cation crosslinking treatment on the active scaffold; transferring the active scaffold subjected to the multivalent cation crosslinking treatment into a polylysine solution, and performing electrostatic complexation treatment to obtain a stable structure; and (3) chelating multivalent cations in the active scaffold subjected to the electrostatic complexation treatment to dissolve out alginate in the active scaffold. The three-dimensional scaffold has a communicated hollow structure, has strong operability, can meet the physical characteristics of stable and specific three-dimensional structures and the like required by biological printing scaffold materials, can carry various cells, medicines and bioactive factors through the communicated hollow structure, is beneficial to nutrition transportation and exchange, is beneficial to accelerating tissue engineering repair, and has good structural stability and excellent controllability of pores.
Description
Technical Field
The invention relates to the technical field of tissue engineering repair, in particular to a three-dimensional stent with a communicated hollow structure and a preparation method thereof.
Background
Tissue engineering is to construct a material complex with bioactivity at the cellular level and molecular level by using the principles and methods of engineering and life science, and the complex can permanently replace and replace tissues and organs with defects or dysfunction in form, structure and function, thereby realizing the functional reconstruction and regeneration of the tissues and organs. In recent years, 3D printing has received much attention from researchers in the field of tissue engineering as an emerging technology. As an important branch of the 3D printing technology, the biological 3D printing enables cells to grow and develop in a suitable material microenvironment for a long time by performing accurate three-dimensional space layout on biological materials, cells and bioactive factors, realizes bionic construction of human tissues and organs, and provides a brand-new strategy for personalized treatment of the tissues and organs.
Alginate (Alg) and Polylysine (PL) are material combinations that have been widely used in the fields of biomedicine and tissue engineering in recent years. The traditional alginate and polylysine systems focus on encapsulating cells in the form of microspheres or microcapsules, and treating chronic diseases such as diabetes, central nervous system diseases and cardiovascular diseases after being implanted into the body. However, such free microspheres or microcapsules lacking in binding effect with each other are difficult to form a specific three-dimensional geometric shape and structure, have poor pore controllability, and have limited application in the construction of three-dimensional bionic tissue organs (e.g., bone repair) in tissue engineering.
Due to the characteristic of rapid ionic crosslinking, alginate is widely researched in the field of bioprinting, and the basic process comprises the following steps: mixing alginate with the cell suspension to obtain biological ink, and extruding the biological ink and performing real-time crosslinking; or mixing a multivalent cation solution and the like in the biological ink for pre-crosslinking, thereby maintaining the stable structure of the extruded ink.
Alginate bioprinting scaffolds of ionically crosslinked systems, however, suffer from structural instability, such as structural failure with loss of multivalent cations. Although the bioprinting effect of alginate can be improved by chemically modifying alginate (such as grafting photocrosslinking groups) or compounding alginate with other materials with stable crosslinking modes, the simplicity and convenience of the preparation process can be significantly influenced. Although there is no multivalent ion alginic acid 3D printing system reported, and a scaffold with a stable structure can be obtained by amidating carboxyl groups of alginate and amino groups of polylysine with EDC/NSH chemical crosslinking solution, the survival rate of cells is greatly reduced by excessively high ink viscosity and EDC/NSH chemical crosslinking treatment, so that it is difficult to achieve cell-loaded bioprinting. In addition, most of the traditional alginate biological printing supports are solid structures, the diffusion resistance of substances is large, the internal nutrition environment is not beneficial to cell growth, and how to endow the alginate biological printing supports with more possibilities in design is also the direction of research and exploration at present, such as the formation of a communicated hollow structure beneficial to nutrition conveying and exchange.
Disclosure of Invention
In view of the defects in the prior art, the invention provides the three-dimensional stent with the communicated hollow structure and the preparation method thereof, which can meet the physical characteristics of stable and specific three-dimensional structures and the like required by biological printing stent materials, also obtain the three-dimensional stent with the communicated hollow structure, endow more possibilities on the design of alginate biological printing stents and facilitate the transportation and exchange of nutrition.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing a three-dimensional scaffold with a communicating hollow structure comprises the following steps:
compounding cells and alginate hydrogel to prepare 3D printing biological ink;
constructing an active scaffold with a three-dimensional structure through 3D printing;
carrying out multivalent cation crosslinking treatment on the active scaffold to obtain a relatively stable structure;
transferring the active scaffold subjected to the multivalent cation crosslinking treatment into a polylysine solution, and performing electrostatic complexation treatment to obtain a stable structure;
chelating multivalent cations in the active scaffold after the electrostatic complexing reaction treatment, so that alginate in the active scaffold is dissolved out.
As one embodiment, before the step of constructing the active scaffold having a three-dimensional structure by 3D printing, the method further comprises: and mixing a multivalent cation solution in the bio-ink to perform pre-crosslinking.
In one embodiment, the step of chelating multivalent cations inside the active scaffold to dissolve out alginate inside the active scaffold comprises:
and (3) putting the active bracket into a sodium citrate solution or an ethylene diamine tetraacetic acid solution for hollowing treatment, wherein the treatment time is 3-20 min.
As one embodiment, the method further comprises, after the electrostatic complexation treatment and before the chelation of the multivalent cations inside the active scaffold:
and (3) placing the active stent subjected to the electrostatic complexation treatment into an alginate solution for structure stabilization treatment, wherein the treatment time is 3-20 min.
As one embodiment, the preparation process of the alginate hydrogel comprises the following steps:
dissolving alginate in phosphate buffer solution, stirring uniformly to obtain alginate hydrogel with the mass fraction of 1-20%, and adjusting the pH value of the alginate hydrogel to 6.5-7.5.
As one of the embodiments, the preparation process of the alginate solution subjected to the structure stabilization treatment comprises:
dissolving alginate in phosphate buffer solution, stirring uniformly to obtain alginate solution with mass fraction of 1-10%, and adjusting pH value of the alginate solution to 6.5-7.5.
In one embodiment, the polylysine solution is replaced with one or more of polyvinylamine, poly-L-ornithine, polymethylene-coguanidine.
In one embodiment, the multivalent cation crosslinking agent used for the multivalent cation crosslinking treatment of the active scaffold is selected from calcium chloride, calcium sulfate or a non-calcium ion multivalent cation crosslinking agent.
The invention also aims to provide a three-dimensional stent with a communicated hollow structure, which is prepared by adopting the preparation method of the three-dimensional stent with the communicated hollow structure.
According to the invention, the biological ink with certain viscosity is subjected to multivalent cation crosslinking treatment after being printed, and is subjected to electrostatic complexation reaction, and finally, the multivalent cation is chelated, so that the formed three-dimensional scaffold has a communicated hollow structure, the preparation process has strong operability, the physical characteristics such as stable and specific three-dimensional structure required by a biological printing scaffold material are met, and besides, various cells, medicines and bioactive factors can be carried by the communicated hollow structure, so that nutrition transportation and exchange are facilitated, the period of tissue engineering repair is favorably shortened, the structure stability of the communicated three-dimensional scaffold with the hollow structure is good, and the controllability of pores is excellent.
Drawings
FIG. 1 is a flow chart of a method of preparing a three-dimensional scaffold according to an embodiment of the present invention;
FIG. 2 is a diagram of a three-dimensional stent with interconnected hollow structures according to an embodiment of the present invention;
FIG. 3 is a partial detailed view of a three-dimensional stent having interconnected hollow structures according to an embodiment of the present invention;
FIG. 4 is a diagram illustrating the effect of dying and alive cells of a three-dimensional scaffold with a connected hollow structure according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Referring to fig. 1, an embodiment of the present invention provides a method for preparing a three-dimensional scaffold, including:
and S01, compounding the cells and alginate hydrogel to prepare the 3D printing biological ink.
S02, constructing an active scaffold with a three-dimensional structure through 3D printing;
s03, carrying out multivalent cation crosslinking treatment on the active scaffold to obtain a relatively stable structure;
s04, transferring the active scaffold subjected to the multivalent cation crosslinking treatment into a polylysine solution, and performing electrostatic complexation treatment to obtain a stable structure;
s05, placing the active scaffold subjected to the electrostatic complexation treatment into an alginate solution for structure stabilization treatment, and adsorbing alginate on the surface of the active scaffold again;
and S06, chelating multivalent cations in the active scaffold after the electrostatic complexing reaction treatment, so that alginate in the active scaffold is dissolved out to form a three-dimensional scaffold with a hollow structure.
Here, in the actual operation process, whether to perform step S05 can be selected according to the application requirements to determine whether to adsorb alginate again on the active scaffold surface.
Before 3D printing is carried out in step S02, calcium solution can be mixed in the bio-ink for pre-crosslinking according to actual conditions so as to reduce the concentration of alginate.
In this embodiment, the polylysine solution may be replaced by one or more of polyethylene imine (polyvinylamine), poly-L-ornithtine (poly-L-ornithine), and polyethylene-co-guanidine (polymethylene-co-guanidine).
The three-dimensional scaffold with the hollow structure prepared by the preparation method not only meets the physical characteristics such as stable and specific three-dimensional structure required by a bioprinting scaffold material, but also can carry various cells, medicines and bioactive factors by communicating the hollow structure, is beneficial to nutrition transportation and exchange, is beneficial to accelerating the period of tissue engineering repair, gives more possibilities to the design of the alginate bioprinting scaffold, and has strong operability in the whole preparation process.
Before the preparation of the three-dimensional scaffold, a preliminary material may be prepared, and the preliminary material may specifically include:
(1) Preparing alginate hydrogel.
When preparing the alginate hydrogel, specifically, the alginate may be dissolved in PBS (phosphate buffer solution), and after stirring uniformly, the alginate hydrogel with a mass fraction of 1% to 20% is prepared, and the pH value of the alginate hydrogel is adjusted to 6.5 to 7.5.
(2) Preparing the multivalent cation crosslinking agent, wherein the multivalent cation crosslinking agent is preferably calcium chloride solution.
In other embodiments, the multivalent cation crosslinker can be calcium sulfate solution or otherwise, or other non-calcium ion multivalent cation (e.g., strontium, barium, etc.) crosslinker (e.g., barium chloride, strontium chloride, etc.).
In this embodiment, the preparation method is to dissolve calcium chloride in PBS, and to prepare a calcium chloride solution with a molar concentration of 90 to 120nmol/L after stirring uniformly.
(3) A polylysine solution was prepared.
Firstly, polylysine is dissolved in PBS, and after stirring uniformly, a polylysine solution with a mass fraction of 2% -15% is prepared, and the pH value of the polylysine solution is adjusted to 6.5-7.5.
(4) An alginate solution was prepared.
The alginate solution is used for structural stabilization, and the preparation process comprises the steps of firstly dissolving alginate in phosphate buffer solution, uniformly stirring to obtain 1-10% by mass of alginate solution (preferably 2-8%), and adjusting the pH value of the alginate solution to 6.5-7.5, wherein in the embodiment, the preparation environment is selected at room temperature.
After the preparation of the early-stage material is completed, the following early-stage preparation of the stent can be carried out:
(1) And preparing the 3D printing biological ink.
Specifically, at room temperature, mixing a certain amount of cells and alginate hydrogel, and uniformly stirring to obtain the 3D printing biological ink with bioactivity.
(2) And (7) printing a support.
Specifically, at room temperature, bioactive ink is filled into a printing cylinder, the extrusion pressure is set to be 20-100Kpa, and fibers extruded from the needle of the cylinder are overlapped layer by layer to form an active scaffold with a porous three-dimensional structure.
After the earlier stage preparation of the support is completed, the following post-treatment of the support is carried out:
(1) And (4) crosslinking treatment.
And (3) immersing the printed active scaffold carrying the cells into a calcium chloride solution for crosslinking at room temperature so as to enhance the structural stability of the scaffold. The treatment time of the step is 3-20 min, and the treated product is washed by PBS, and generally washed for three times.
(2) And (4) carrying out electrostatic complexation treatment.
And (3) at room temperature, placing the active scaffold crosslinked by the multivalent cations into a polylysine solution for carrying out electrostatic complexation treatment for 3-20 min, and washing the active scaffold with PBS (phosphate buffered saline) after the treatment, wherein the washing is generally carried out for three times.
(3) And (5) carrying out structural stabilization treatment.
And (3) at room temperature, putting the polylysine subjected to electrostatic complexation into an alginate solution for structural stabilization, wherein the treatment time is 3-20 min, and washing the active scaffold with PBS (phosphate buffer solution) for three times.
(4) And (5) hollowing.
And (3) putting the active scaffold with the stable structure into a sodium citrate solution at room temperature for hollowing treatment for 3-20 min, and washing with PBS (phosphate buffer solution) after treatment, wherein the washing is generally carried out for three times. Thus, the multivalent cations in the active scaffold are chelated, and the alginate in the active scaffold is dissolved out.
As shown in FIGS. 2 and 3, the three-dimensional scaffold with a communicated hollow structure for loading cells can be obtained by the technical scheme. As shown in FIG. 4, the staining effect of the three-dimensional scaffold with interconnected hollow structure is shown, and the white dots in the figure represent the cells, and most of the cells are in a viable state. The treatment time of the alginate solution, the calcium chloride solution, the polylysine solution and the sodium citrate solution can be properly adjusted according to the size of the stent fiber.
Wherein, the alginate can be sodium alginate and other hydrogels which can be quickly cross-linked by multivalent cations, such as materials with the same side chain carboxyl group and negative charge property as the alginate; polylysine can be artificially synthesized or biosynthesized, and can also be other materials with positive charge characteristics, such as chitosan, chitosan oligosaccharide and the like. In other embodiments, citric acid may be substituted with other agents that can sequester multivalent cations, including EDTA. The alginate hydrogel can be loaded with various cells, medicines and bioactive factors without damaging the structure. When the load is loaded with drugs or bioactive factors, the PBS can be replaced by deionized water to treat the whole process or part of the process.
The three-dimensional scaffold prepared by the method meets the physical characteristics of stable and specific three-dimensional structures and the like required by biological printing scaffold materials, and can carry various cells, medicines and bioactive factors through the communicated hollow structure, so that nutrition transportation and exchange are facilitated, and the period of tissue engineering repair is accelerated. Most of the traditional alginate biological printing supports are solid structures, and the diffusion resistance of substances is large, so that the internal nutrient environment is not favorable for the growth and development of cells; the three-dimensional support communicated with the hollow structure has good structural stability and excellent controllability of pores. Compared with the prior art, the free microspheres or microcapsules which lack the binding effect with each other cannot form a specific three-dimensional stable structure meeting the requirement of tissue regeneration and repair, and the preparation process of the three-dimensional scaffold has strong operability.
The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.
Claims (7)
1. A method for preparing a three-dimensional scaffold with a communicated hollow structure is characterized by comprising the following steps:
compounding cells and alginate hydrogel to prepare 3D printing biological ink;
constructing an active scaffold with a three-dimensional structure through 3D printing;
carrying out multivalent cation crosslinking treatment on the active scaffold to obtain a relatively stable structure;
transferring the active scaffold subjected to the multivalent cation crosslinking treatment into a polylysine solution, and performing electrostatic complexation treatment to obtain a stable structure;
chelating multivalent cations in the active scaffold after the electrostatic complexing reaction treatment, so that alginate in the active scaffold is dissolved out;
wherein, the ionic crosslinking agent used for carrying out the multivalent cation crosslinking treatment on the active scaffold is selected from calcium chloride or calcium sulfate cationic crosslinking agent; the step of chelating multivalent cations inside the active scaffold to dissolve out alginate inside the active scaffold comprises the following steps: and (3) putting the active bracket into a sodium citrate solution or an ethylene diamine tetraacetic acid solution for hollowing treatment, wherein the treatment time is 3-20 min.
2. The method for preparing a three-dimensional scaffold with interconnected hollow structures according to claim 1, wherein the step of constructing an active scaffold with three-dimensional structures by 3D printing is preceded by the further steps of: and mixing a multivalent cation solution in the bio-ink to perform pre-crosslinking.
3. The method for preparing a three-dimensional scaffold with interconnected hollow structures according to claim 1, further comprising, after the electrostatic complexation treatment and before chelating multivalent cations inside the active scaffold:
and (3) placing the active scaffold subjected to the electrostatic complexation treatment into an alginate solution for structural stabilization treatment, wherein the treatment time is 3-20 min.
4. The method for preparing the three-dimensional scaffold with the interconnected hollow structures as claimed in claim 1, wherein the alginate hydrogel is prepared by a process comprising:
dissolving alginate in phosphate buffer solution, stirring uniformly to obtain alginate hydrogel with the mass fraction of 1-20%, and adjusting the pH value of the alginate hydrogel to 6.5-7.5.
5. The method for preparing a three-dimensional scaffold having interconnected hollow structures according to claim 3, wherein the preparation of the alginate solution for the structure-stabilizing treatment comprises:
dissolving alginate in phosphate buffer solution, stirring uniformly to obtain alginate solution with mass fraction of 1% -10%, and adjusting the pH value of the alginate solution to 6.5-7.5.
6. The method for preparing a three-dimensional scaffold with an interconnected hollow structure according to any one of claims 1 to 5, wherein the polylysine solution is replaced by one or more of polyvinylamine, poly-L-ornithine and polymethylene-coguanidine.
7. A three-dimensional scaffold having interconnected hollow structures, which is prepared by the method for preparing a three-dimensional scaffold having interconnected hollow structures according to any one of claims 1 to 6.
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| CN113893387B (en) * | 2021-09-09 | 2023-02-03 | 大连理工大学 | Tissue engineering scaffold assembled by cell-loaded microgel and preparation method and application thereof |
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| CN102101037B (en) * | 2009-12-18 | 2013-09-18 | 中国科学院大连化学物理研究所 | Preparation method of alginate/epsilon-polylysine/alginate biological microcapsule |
| CN106944166A (en) * | 2017-04-01 | 2017-07-14 | 太原理工大学 | A kind of method that use biometric print prepares microfluidic channel model |
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