CN111704727A - Canaglucose/polydopamine hybrid hydrogel and preparation method and application thereof - Google Patents

Canaglucose/polydopamine hybrid hydrogel and preparation method and application thereof Download PDF

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CN111704727A
CN111704727A CN202010492056.9A CN202010492056A CN111704727A CN 111704727 A CN111704727 A CN 111704727A CN 202010492056 A CN202010492056 A CN 202010492056A CN 111704727 A CN111704727 A CN 111704727A
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polydopamine
hydrogel
polysaccharide
hybrid hydrogel
scaffold
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CN111704727B (en
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齐晓亮
童显琴
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Wenzhou Medical University
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    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • 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
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    • A61L27/14—Macromolecular materials
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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
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    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/06—Polycondensates having nitrogen-containing heterocyclic rings in the main chain of the macromolecule
    • C08G73/0666—Polycondensates containing five-membered rings, condensed with other rings, with nitrogen atoms as the only ring hetero atoms
    • C08G73/0672—Polycondensates containing five-membered rings, condensed with other rings, with nitrogen atoms as the only ring hetero atoms with only one nitrogen atom in the ring
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    • C08J2479/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen, or carbon only, not provided for in groups C08J2461/00 - C08J2477/00
    • C08J2479/04—Polycondensates having nitrogen-containing heterocyclic rings in the main chain; Polyhydrazides; Polyamide acids or similar polyimide precursors

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Abstract

The invention relates to a method for preparing a hybrid hydrogel of polysaccharide and polydopamine and application thereof, wherein the preparation method comprises the following steps: the polydopamine solution is prepared by oxidative self-polymerization under a certain condition, the obtained natural polysaccharide and the polydopamine are uniformly dispersed, heated to 80 ℃ and then cooled to form hydrogel, the hydrogel is co-cultured with various fibroblasts to show good biocompatibility, the polydopamine can regulate and control the microstructure, the mechanical property, the swelling property, the ion response behavior and the like of the obtained natural polysaccharide hydrogel, so that the hybrid hydrogel can be used as a tissue engineering material for in-vitro culture of the fibroblasts, and an in-vivo application mode of the polysaccharide-based hydrogel in tissue engineering is provided.

Description

Canaglucose/polydopamine hybrid hydrogel and preparation method and application thereof
Technical Field
The invention relates to the field of biological scaffolds, in particular to a natural polysaccharide/polydopamine hybrid hydrogel and a preparation method and application thereof.
Background
Tissue engineering is an emerging discipline combining principles and methods of engineering and life sciences. Scientists have been working on developing three-dimensional scaffold materials that better mimic cells as an environment. An ideal cell scaffold should have good biocompatibility, degradability, three-dimensional porous structure, certain biomechanical properties, and provide a good cell interface. The biomechanical property and chemical environment of the cell scaffold material are two important factors influencing the biological behavior of cells.
The selection and optimization of scaffold materials is an especially important link in tissue engineering. Current scaffold materials are mainly classified into synthetic macromolecules and natural biomaterials. The polysaccharide is a natural polysaccharide of bacterial origin, which has been widely used in food chemistry due to its excellent biocompatibility, and scholars have published applications for delivery of small molecule drugs, proteins, genes, etc. Polydopamine is a natural biological material of mussel, and has the functions of removing oxygen free radicals, promoting osteogenesis and the like.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a natural polysaccharide/polydopamine hybrid hydrogel as well as a preparation method and application thereof.
In order to achieve the purpose, the invention provides a preparation method of a natural polysaccharide/polydopamine hybrid hydrogel, dopamine monomer is dissolved in sodium hydroxide alkaline solution with the pH value of 13, the solution is stirred for 12 hours at the temperature of 25 ℃, the pH value is adjusted to 8.5, the polydopamine mother solution with the final concentration of 1-10mg/mL is prepared, the natural polysaccharide is dissolved in alkaline solution with the pH value of 12, the final concentration is 30mg/mL, all components are uniformly dispersed, the solution is heated to 80 ℃ and then cooled to form the hydrogel, and finally the polysaccharide-based hybrid hydrogel is obtained.
Further, the final concentration of the polydopamine mother liquor is 1-5 mg/mL.
The invention also provides the native polysaccharide/polydopamine hybrid hydrogel prepared by the preparation method of the native polysaccharide/polydopamine hybrid hydrogel.
The invention also provides application of the native polysaccharide/polydopamine hybrid hydrogel, and application of the native polysaccharide/polydopamine hybrid hydrogel in preparation of cell scaffolds.
As an application mode of the invention, the cell scaffold is a 3D cell culture scaffold.
As an application mode of the invention, the cell scaffold has pores with the diameter of 10-100 μm.
As an application mode of the invention, the cell scaffold is a fibroblast culture scaffold.
As an application mode of the invention, the cell scaffold is a cell culture scaffold with an ion concentration response regulation type, and the ion is one of sodium ion, magnesium ion, aluminum ion or calcium ion.
As an application mode of the invention, the cell scaffold is a calcium ion concentration response regulation type bone cell culture scaffold.
The invention has the following advantages: the physical gelling property of the natural polysaccharide avoids the use of a toxic cross-linking agent, the whole preparation process is simple, the natural biocompatibility is very high, the natural advantage of the natural polysaccharide serving as a cell scaffold is achieved, polydopamine is a mussel-like natural biological material, the adhesive property of the material can be obviously enhanced, and the physical and chemical properties, including mechanical property and swelling property, of the hydrogel can be regulated and controlled by the polydopamine in the preparation process of the natural polysaccharide/polydopamine hybrid hydrogel.
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FIG. 1 is a scanning electron microscope image of an embodiment of the present invention;
FIG. 2 is a graph showing the rheological results of hydrogels according to examples of the present invention;
FIG. 3 shows the results of swelling performance of hydrogels according to examples of the present invention;
FIG. 4 is a photograph showing the cytotoxicity results of the hydrogel of periodontal ligament cells (hPDLCs) in the examples of the present invention;
FIG. 5 is a photograph of live and dead cells of a hydrogel of periodontal ligament cells (hPDLCs) in an example of the present invention;
FIG. 6 shows the result of cytotoxicity of mouse fibroblasts (L929) in the hydrogel of the present invention.
Detailed Description
The present invention will be further described in detail with reference to examples and effect examples, but the scope of the present invention is not limited thereto.
Example 1: preparation of hybrid hydrogel of polysaccharide/polydopamine
The preparation method comprises the following steps:
dissolving dopamine monomer in sodium hydroxide alkaline solution with pH value of 13, stirring for 12h at 25 ℃, adjusting pH to 8.5, preparing polydopamine mother liquor with final concentration of 1mg/mL, dissolving polysaccharide in alkaline solution with pH value of 12 with final concentration of 30mg/mL, mixing, uniformly dispersing all components, heating to 80 ℃, and cooling to form hydrogel.
Example 2: preparation of hybrid hydrogel of polysaccharide/polydopamine
The preparation method comprises the following steps:
dissolving dopamine monomer in sodium hydroxide alkaline solution with pH value of 13, stirring for 12h at 25 ℃, adjusting pH to 8.5, preparing polydopamine mother liquor with final concentration of 2mg/mL, dissolving polysaccharide in alkaline solution with pH value of 12 with final concentration of 30mg/mL, mixing, uniformly dispersing all components, heating to 80 ℃, and cooling to form hydrogel.
Example 3: preparation of hybrid hydrogel of polysaccharide/polydopamine
The preparation method comprises the following steps:
dissolving dopamine monomer in sodium hydroxide alkaline solution with pH value of 13, stirring for 12h at 25 ℃, adjusting pH to 8.5, preparing polydopamine mother liquor with final concentration of 4mg/mL, dissolving polysaccharide in alkaline solution with pH value of 12 with final concentration of 30mg/mL, mixing, uniformly dispersing all components, heating to 80 ℃, and cooling to form hydrogel.
Example 4: morphological characteristics of native polysaccharide/polydopamine hybrid hydrogel
The evaluation steps are as follows:
setting a control group, dissolving the polysaccharide in an alkaline solution with the pH value of 12 to obtain a final concentration of 30mg/mL, heating to 80 ℃, and cooling to form water condensate, namely CP 0; the hydrogels prepared in examples 1-3 were designated CP1, CP2, and CP3, respectively.
Soaking CP0, CP1, CP2 and CP3 hydrogel in deionized water for 12h to reach swelling balance, quickly freezing with liquid nitrogen, freeze-drying the sample with a freeze dryer for 12h, spraying gold, and observing with a scanning electron microscope.
The results in fig. 1 show that as the concentration of polydopamine increases, the pore size of the hybrid hydrogel gradually decreases. The polydopamine has a regulating effect on the microstructure of the hydrogel scaffold.
Example 5: rheological performance results of the native polysaccharide/polydopamine hybrid hydrogel
The evaluation steps are as follows:
setting a control group, dissolving the polysaccharide in an alkaline solution with the pH value of 12 to obtain a final concentration of 30mg/mL, heating to 80 ℃, and cooling to form water condensate, namely CP 0; the hydrogels prepared in examples 1-3 were designated CP1, CP2, and CP3, respectively.
The loss modulus was measured using a rheometer with CP0, CP1, CP2, and CP3 hydrogels. The results in fig. 2 show that as the concentration of polydopamine increases, the mechanical properties of the gel are gradually enhanced and optimized, and the concentration of polydopamine is correlated, and when in application, the gels with different mechanical properties are selected according to different use environments.
Example 6: the swelling performance results of the native polysaccharide/polydopamine hybrid hydrogel were obtained
The evaluation steps are as follows:
setting a control group, dissolving the polysaccharide in an alkaline solution with the pH value of 12 to obtain a final concentration of 30mg/mL, heating to 80 ℃, and cooling to form water condensate, namely CP 0; the hydrogel prepared in example 3 was designated CP 3.
CP0, CP3 hydrogels were soaked in deionized water to a swelling equilibrium and the initial mass was measured at time points with the mass at each corresponding time point. According to the formula: (Mt-M0)/M0; mt is the weight at the corresponding time point, and M0 is the initial mass.
The CP0 and CP3 hydrogels were taken and subjected to swelling experiments again in 1.5% sodium chloride, magnesium chloride and aluminum chloride solutions, respectively. Higher swelling more readily leads to collapse of the three-dimensional scaffold, and the results in fig. 3 show that the hybrid hydrogel exhibits lower swelling properties and the system is more stable than the native polysaccharide hydrogel. Also, the swelling properties show differences in the sodium magnesium aluminum ion solution, exhibiting ion response behavior. The strong interaction between polydopamine and metal ions compresses the structure, and the hydrogel network is further reduced.
The hydrogel exhibits responsive swelling behavior in ions of different valency states. Ions with different valence states and a polydopamine chain are subjected to secondary crosslinking, so that the winding tightness of the hydrogel polymer chain is changed, and the physical and chemical properties and the like are correspondingly changed. Therefore, the hydrogel scaffold constructed by the invention can regulate and improve the physical and chemical properties of the gel by changing the valence state, concentration and the like of ions, and becomes a matrix more suitable for cell culture: when the hydrogel cell is used as an osteoblast scaffold, the adhesion and differentiation of osteoblasts can be regulated by adsorbing/releasing calcium ions.
Example 7: evaluation result of biocompatibility of natural polysaccharide/polydopamine hybrid hydrogel
The evaluation steps are as follows:
setting a control group, dissolving the polysaccharide in an alkaline solution with the pH value of 12 to obtain a final concentration of 30mg/mL, heating to 80 ℃, and cooling to form water condensate, namely CP 0; the hydrogel prepared in example 3 was designated CP 3.
Preparing gel samples of CP0 and CP3 hydrogel, preparing leachate with the volume ratio of 1: 1, taking out the leachate, and adding fetal calf serum with the volume ratio of 1: 10.
Human periodontal ligament cells were plated in 96-well plates at a density of 5 × 103one/mL. After overnight culture, CP0 and CP3 extracts were added for co-culture, and human periodontal ligament cells were cultured directly in fetal bovine serum plated 96-well plates with a blank.Cell proliferation activity was tested on days 1, 3, 5 using CCK-8 kit human pericyte cells plated on laser confocal dish at density 5 × 103one/mL. After overnight culture, adding CP0 and CP3 leachate for co-culture, staining with Calcein-AM/PI alive staining agent for 1, 3, 5 days, and observing by laser confocal observation. The results in fig. 4 show that the 1, 3 and 5-day hybrid hydrogel has at least 80% of cell activity relative to the control group and the CP0 group, and fig. 5 shows that the fluorescence staining is performed with the most living cells, the dead cells are not basically seen, the cell morphology presents fusiform spindle-shaped fibroblasts, and the preparation method has low biological toxicity and high biocompatibility to human periodontal ligament cells.
Mouse fibroblasts (L929) were additionally plated in 96-well plates at a density of 104one/mL. After overnight incubation, CP0 was added and CP3 extract was co-incubated. The cell proliferation activity was tested using CCK-8 kit at 1, 3, 5 days, and the results are shown in fig. 6, and the native polysaccharide/polydopamine hybrid hydrogel can promote the proliferation of fibroblasts.
Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims (9)

1. A preparation method of a native polysaccharide/polydopamine hybrid hydrogel is characterized by comprising the following steps: dissolving dopamine monomer in sodium hydroxide alkaline solution with pH value of 13, stirring for 12h at 25 ℃, adjusting pH to 8.5, preparing polydopamine mother liquor with final concentration of 1-10mg/mL, dissolving the obtained polysaccharide in alkaline solution with pH value of 12 with final concentration of 30mg/mL, uniformly dispersing all components, heating to 80 ℃, and cooling to form hydrogel, thereby finally obtaining the polysaccharide-based hybrid hydrogel.
2. A preparation method of a native polysaccharide/polydopamine hybrid hydrogel is characterized by comprising the following steps: the final concentration of the polydopamine mother liquor is 1-5 mg/mL.
3. A hybrid hydrogel of polysaccharide/polydopamine, wherein the gel is prepared by the method according to claim 1 or 2.
4. Use of a hybrid hydrogel of a polysaccharide/polydopamine according to claim 3 for the preparation of a cytoskeleton.
5. The use of the hybrid hydrogel according to claim 4, wherein the cell scaffold is a 3D cell culture scaffold.
6. The use of a hybrid hydrogel according to claim 4, wherein said cell scaffold has pores with a diameter of 10-100 μm.
7. The use of the hybrid hydrogel according to claim 4, wherein the cell scaffold is a fibroblast culture scaffold.
8. The use of the hybrid hydrogel according to claim 7, wherein the cell scaffold is a cell culture scaffold with a controlled response to ion concentration, and the ion is one of sodium ion, magnesium ion, aluminum ion, or calcium ion.
9. The use of the hybrid hydrogel according to claim 8, wherein the cell scaffold is a calcium ion concentration response-controlled bone cell culture scaffold.
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CN113384756A (en) * 2021-06-22 2021-09-14 山东大学 Chitosan composite scaffold material for in-situ loading of polydopamine and preparation method thereof
CN113845668A (en) * 2021-08-06 2021-12-28 广东省科学院健康医学研究所 Polysaccharide composite hydrogel and preparation method and application thereof

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CN113004544A (en) * 2021-03-03 2021-06-22 中科院过程工程研究所南京绿色制造产业创新研究院 Agarose hydrogel and preparation method and application thereof
CN113384756A (en) * 2021-06-22 2021-09-14 山东大学 Chitosan composite scaffold material for in-situ loading of polydopamine and preparation method thereof
CN113845668A (en) * 2021-08-06 2021-12-28 广东省科学院健康医学研究所 Polysaccharide composite hydrogel and preparation method and application thereof
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