CN116251232B - Bone tissue repair composite scaffold and preparation method and application thereof - Google Patents
Bone tissue repair composite scaffold and preparation method and application thereof Download PDFInfo
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Classifications
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- 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
- A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention belongs to the technical field of functional materials, and particularly relates to a bone tissue repair composite scaffold, and a preparation method and application thereof. The invention utilizes electrostatic interaction between aldehyde sodium alginate and gelatin, adopts a layer-by-layer self-assembly method to respectively cover IL-4 and IFN-gamma cytokines on the inner layer and the outer layer of gelatin-aldehyde sodium alginate double-layer microdroplet liquid, adopts a directional heat induction assembly method to construct a collagen-nano hydroxyapatite bracket with a plate layered structure, and then adopts a liquid-liquid phase separation method to deposit gelatin-aldehyde sodium alginate double-layer microdroplet liquid which covers the IL-4 and IFN-gamma cytokines in the inner space structure of the collagen-nano hydroxyapatite bracket, thereby being capable of sequentially and timely releasing the IL-4 and IFN-gamma cytokines in a bone defect area, accurately regulating and controlling immune reaction of the bone defect area, orderly activating M1 macrophages and M2 macrophages, releasing vascular related factors VEGF and PEGF-BB, and realizing endogenous regeneration of blood vessels.
Description
Technical Field
The invention belongs to the technical field of functional materials, and particularly relates to a bone tissue repair composite scaffold, and a preparation method and application thereof.
Background
Collagen and nano hydroxyapatite are main organic/inorganic components of human bones, have good biological activity and biocompatibility, and are well-known excellent bone tissue repair materials. Whether or not the scaffold material undergoes angiogenesis during bone repair determines the success or failure of bone defect repair. The immune system has positive regulation effect on the bone repair process, and macrophages are recruited to the damaged part in the early healing period of bone injury to start an immune mechanism to participate in and mediate the vascularization process of bone defect. Wherein M1 macrophages initiate angiogenesis, and M2 macrophages promote vascular development and maturation. Therefore, the method constructs an effective treatment strategy capable of accurately and dynamically mediating the sequence phenotype polarization of macrophages and promoting the endogenous regeneration of blood vessels, and is used for repairing bone defects.
Macrophages have plasticity and they are able to activate different polarization states in response to signals provided by the biochemical and structural microenvironment of the bone defect region. Activation of macrophages of different phenotypes, in turn, affects the secretion of angiogenesis-related factors, thereby promoting angiogenesis in bone tissue repair scaffolds. Macrophages are polarized towards the M1 type under the mediation of cytokines such as LPS, IFN- γ, TNF- α and IL-1β, and M1 macrophages play an important role in the early stages of angiogenesis, and their secreted inflammatory cytokines TNF- α and IL-1β activate the tip cells, secrete Vascular Endothelial Growth Factor (VEGF), thereby promoting vascular endothelial cell proliferation and recruiting perivascular cells (T.A.Wynn, A.Chawla, J.W.Pollard, macrophage biology in development, homeostasis and disease, nature496 (7446) (2013) 445-455.). M2 macrophages are activated by cytokines such as IL-4, IL-10, IL-13 and TGF-beta, secrete and express high levels of platelet derived growth factor-BB (PDGF-BB), recruit perivascular and mesenchymal stem cells to remodel the initial vascular buds, stabilize vascular growth (A.N.Stratman, A.E.Schwindt, K.M.Malotte, G.E.Davis, endothelial-modified PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization, blood 116 (22) (2010) 4720-4730.). In the prior art, there is still a lack of a bone tissue repair scaffold capable of activating M1 macrophages and M2 macrophages sequentially.
Disclosure of Invention
The first object of the present invention is to provide a bone tissue repair composite scaffold, the second object of the present invention is to provide a method for preparing the same, and the third object of the present invention is to provide an application of the same.
According to a first aspect of the present invention, there is provided a bone tissue repair composite scaffold comprising a gelatin-hydroformylation sodium alginate bilayer droplet encapsulating IL-4 and IFN- γ cytokines supported on an oriented lamellar collagen-nanohydroxyapatite scaffold, wherein the inner layer of the gelatin-hydroformylation sodium alginate bilayer droplet encapsulating IL-4 and IFN- γ cytokines encapsulates IL-4 cytokines and the outer layer encapsulates IFN- γ cytokines.
In some embodiments, the preparation method of the gelatin-formylated sodium alginate bilayer microdroplet coated with IL-4 and IFN-gamma cytokines comprises the following steps:
dissolving aldehyde sodium alginate in PBS buffer solution to obtain aldehyde sodium alginate solution, dissolving gelatin in PBS buffer solution to obtain gelatin solution, and uniformly mixing the aldehyde sodium alginate solution and the gelatin solution to obtain gelatin-aldehyde sodium alginate double-layer microdroplet with positive charges on the inner layer and negative charges on the outer layer;
adding the IL-4 cytokine into gelatin-aldehyde sodium alginate double-layer micro-droplet liquid, uniformly mixing, and adsorbing the IL-4 cytokine on the inner layer by utilizing gelatin with positive charges on the inner layer of the gelatin-aldehyde sodium alginate double-layer micro-droplet liquid to obtain gelatin-aldehyde sodium alginate double-layer micro-droplet liquid coated with the IL-4 cytokine;
and adding the IFN-gamma cytokine into the gelatin-hydroformylation sodium alginate double-layer microdroplet wrapped by the IL-4 cytokine, uniformly mixing, and adsorbing the IFN-gamma cytokine on the outer layer by utilizing the negatively charged hydroformylation sodium alginate on the outer layer of the gelatin-hydroformylation sodium alginate double-layer microdroplet to obtain the gelatin-hydroformylation sodium alginate double-layer microdroplet wrapped by the IL-4 cytokine and the IFN-gamma cytokine. Therefore, the electrostatic interaction between the aldehyde sodium alginate and the gelatin is utilized to respectively coat the IL-4 and IFN-gamma cytokines on the inner layer and the outer layer of the gelatin-aldehyde sodium alginate double-layer micro-droplet liquid by adopting a layer-by-layer self-assembly method.
In some embodiments, the method of preparing the aldehyde sodium alginate comprises the steps of:
according to parts by weight, 5-10 parts of sodium alginate are dissolved in 200 parts of water, 50-100 parts of absolute ethyl alcohol is added after stirring uniformly, 5-10 parts of sodium periodate is added after stirring uniformly and is reacted for 12-24 hours at room temperature in a dark place, then 10-20 parts of ethylene glycol is added to neutralize excessive sodium periodate, the reaction is continued for 2-4 hours in a dark place, 5-10 parts of sodium chloride is added after the reaction is finished, 600-1200 parts of ethanol is used for precipitating out oxidized sodium alginate, then the obtained precipitate is added into water for dissolution to obtain a solution, then ethanol is added for precipitating out oxidized sodium alginate in the solution, the dissolving-precipitating process is repeated for 2-5 times to obtain an aldehyde sodium alginate solution, and finally the aldehyde sodium alginate solution is purified and freeze-dried to obtain the aldehyde sodium alginate.
In some embodiments, the sodium alginate has an oxidation degree of 20%.
In some embodiments, the method of purifying the aldehyde sodium alginate solution is: the aldehyde sodium alginate solution is dialyzed by a dialysis bag with the molecular weight cut-off of 3500 until the sodium periodate is completely removed.
In some embodiments, the concentration of the sodium alginate solution is 2-5wt%, the concentration of the gelatin solution is 2-10wt%, and the volume ratio of the sodium alginate solution to the gelatin solution is 1:1.
In some embodiments, the concentration of IL-4 cytokine is 1 μg/mL, the concentration of IFN-gamma cytokine is 1 μg/mL, and the electrostatic interaction time of IL-4 cytokine and IFN-gamma cytokine with gelatin-formylated sodium alginate bilayer droplets is 2h.
In some embodiments, the method of preparing an oriented lamellar collagen-nano hydroxyapatite scaffold comprises the steps of:
dissolving type I collagen in a solvent to prepare a collagen solution, uniformly dispersing nano hydroxyapatite in the collagen solution to obtain a mixed solution, then utilizing unidirectional temperature gradient to induce the mixed solution to directionally assemble an oriented lamellar collagen-nano hydroxyapatite scaffold, freeze-drying the scaffold after solidification, and utilizing glutaraldehyde to steam and crosslink the scaffold to obtain the stable oriented lamellar collagen-nano hydroxyapatite scaffold.
In some embodiments, the solvent is acetic acid or hydrochloric acid solution, the concentration of type i collagen in the collagen solution is 2-4wt%, and the mass ratio of collagen to nano-hydroxyapatite is 1:2.
in some embodiments, the unidirectional temperature gradient is from-196 ℃ to room temperature.
In some embodiments, the scaffold is fumigated with 25wt% glutaraldehyde for crosslinking for 12-24 hours.
According to a second aspect of the present invention, there is provided a method for preparing the above bone tissue repair composite scaffold, comprising the steps of:
soaking the oriented lamellar collagen-nano hydroxyapatite scaffold in gelatin-aldehyde sodium alginate double-layer micro-droplet liquid coated with IL-4 and IFN-gamma cytokines for 12-24 hours, and then taking out the scaffold for freeze drying to obtain the bone tissue repair composite scaffold.
According to the invention, an electrostatic interaction between aldehyde sodium alginate and gelatin is utilized, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) cytokines are respectively coated on the inner layer and the outer layer of gelatin-aldehyde sodium alginate double-layer microdroplet liquid by adopting a layer-by-layer self-assembly method, an oriented lamellar collagen-nano hydroxyapatite bracket is constructed by adopting a directional heat induction assembly method, then gelatin-aldehyde sodium alginate double-layer microdroplet liquid coated with IL-4 and IFN-gamma cytokines is deposited in the inner space structure of the collagen-nano hydroxyapatite bracket by adopting a liquid-liquid phase separation method, so that a bone tissue repair composite bracket is obtained.
In some embodiments, the ratio of the mass volume of the oriented lamellar collagen-nano hydroxyapatite scaffold to the gelatin-formylated sodium alginate bilayer droplet surrounding IL-4 and IFN-gamma cytokines is 0.05 to 0.2g/mL.
According to a third aspect of the present invention, there is provided the use of the above-described composite scaffold for bone tissue repair in the preparation of a tissue engineering material or a bone defect repair material.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the invention, gelatin and aldehyde sodium alginate are combined through electrostatic interaction under mild conditions, so that bioactive factors IL-4 and IFN-gamma can be partitioned and encapsulated, double-layer micro-droplet liquid with good cell compatibility is formed, the inner layer and the outer layer of the double-layer micro-droplet liquid are respectively wrapped with IL-4 and IFN-gamma cytokines for regulating and controlling macrophages, then the double-layer micro-droplet liquid wrapped with the IL-4 and IFN-gamma cytokines is attached to an oriented lamellar collagen-nano hydroxyapatite bracket, the biological response sequential release of the IL-4 and IFN-gamma cytokines can be regulated and controlled by the orientation space structure of the bracket and the design of the double-layer micro-droplet liquid, the M1 and M2 phenotype sequential activation of the macrophages is effectively and timely stimulated, a proper immune micro-environment is triggered, and then the behaviors of the macrophages are regulated and controlled in a space-time mode, so that the aim of accurately regulating the immune performance and thus realizing rapid vascularization is achieved.
(2) The invention adopts the directional self-assembly technology to develop the composite scaffold similar to the natural mature bone tissue composition and the lamellar structure, simulates the natural bone from the composition and the structure, is a good bone tissue repair scaffold, and has wider application prospect in the field of bone repair.
Drawings
Fig. 1 is a schematic structural view of a mold according to embodiment 1 of the present invention.
FIG. 2 is a schematic diagram of the directional induction apparatus according to example 1 of the present invention.
FIG. 3 is a fluorescence image of a droplet, wherein A1-A2 are a microscopic image and a fluorescence image, respectively, of a single layer droplet; B1-B2 are microscopic and fluorescent images of the bilayer droplets, respectively.
FIG. 4 is a scanning electron microscope image of a cross section and a longitudinal section of the Col-I-nHAP stent prepared in example 1.
FIG. 5 is a cross-sectional scanning electron microscope image of the Col-I-nHAP scaffold prepared in examples 1-3, in sequence from left to right.
FIG. 6 is a graph of the porosity of the Col-I-nHAP scaffolds prepared in examples 1-3.
FIG. 7 is a pore distribution diagram of the Col-I-nHAP scaffold prepared in examples 1-3.
Detailed Description
The invention will now be described in further detail with reference to the accompanying drawings, it being understood that the following examples are given for the purpose of better illustration only and are not intended to limit the scope of the invention. The process steps not disclosed in the examples are prior art. Unless otherwise specified, the following materials are all commercially available.
It is to be noted that the term "room temperature" as used herein means 10℃to 30 ℃. In the invention, the polytetrafluoroethylene tube is hollow and the polytetrafluoroethylene rod is solid.
In the embodiment of the invention, the manufacturing method of the die is as follows: as shown in fig. 1, one end of a polytetrafluoroethylene tube 2 with an inner diameter of about 5mm is fixed on a carrier 1, and a solid polytetrafluoroethylene rod 3 with an outer diameter of about 3mm is placed in the center of the polytetrafluoroethylene tube 2, so that an annular mold is obtained; meanwhile, a first space 4 is formed between the polytetrafluoroethylene rod 3 and the polytetrafluoroethylene tube 2; the carrier 1 may be a glass sheet.
The temperature gradient in the invention means that the die is placed at two different temperature points to form gradient temperature, and as shown in figure 2, the lower part of the die is at-196 ℃ and the upper part of the die is at room temperature. Wherein, -196 ℃ is the liquid nitrogen temperature.
In the following examples, sodium alginate was used having an oxidation degree of 20%.
The preparation method of the PBS buffer solution comprises the following steps: 800mL of distilled water was prepared in a vessel, and then 8g of NaCl, 200mg of KCl, 1.44g of Na were sequentially added to the distilled water 2 HPO 4 、240mg KH 2 PO 4 The solution pH was adjusted to 7.4 and distilled water was then added until the solution volume was 1L.
Example 1
The preparation method of the bone tissue repair composite scaffold of the embodiment comprises the following steps:
s1, dissolving 5g of sodium alginate in 200mL of deionized water, stirring uniformly, adding 50mL of absolute ethyl alcohol, stirring uniformly, adding 5.7g of sodium periodate in a dark place, reacting for 24 hours at room temperature in a dark place, adding 10mL of ethylene glycol to neutralize excessive sodium periodate, continuing to react for 2 hours in a dark place, adding 5g of sodium chloride after the reaction is finished, precipitating oxidized sodium alginate with 800mL of ethyl alcohol, adding the obtained precipitate into 100mL of deionized water to dissolve to obtain a solution, adding 600mL of ethyl alcohol to precipitate oxidized sodium alginate in the solution, and repeating the dissolving-precipitating process for 3 times to obtain the hydroformylation sodium alginate solution. Dialyzing the obtained aldehyde sodium alginate solution by using a dialysis bag with the molecular weight cut-off of 3500 until the sodium periodate is completely removed, and finally freeze-drying the dialyzed solution to obtain the aldehyde sodium alginate (OSA for short).
S2, dissolving aldehyde sodium alginate into PBS buffer solution to prepare 2wt% aldehyde sodium alginate solution (abbreviated as OSA solution), dissolving gelatin (abbreviated as GA) into PBS buffer solution to prepare 2wt% gelatin solution (abbreviated as GA solution), and taking equal volumes of OSA solution and GA solution to vortex and mix for 1 minute to obtain gelatin-aldehyde sodium alginate (GA-OSA) double-layer microdroplet liquid with positive charges on the inner layer and negative charges on the outer layer;
adding the concentrated solution of the IL-4 cytokine into the GA-OSA double-layer microdroplet, wherein the concentration of the IL-4 cytokine is 1 mug/mL, magnetically stirring for 2 hours, uniformly mixing, and completely adsorbing the IL-4 cytokine on the inner layer by utilizing gelatin with positive charges on the inner layer of the GA-OSA double-layer microdroplet to obtain the GA-OSA double-layer microdroplet wrapped by the IL-4 cytokine;
and adding the concentrated solution of the IFN-gamma cell factor into the GA-OSA double-layer microdroplet wrapped by the IL-4 cell factor, wherein the concentration of the IFN-gamma cell factor is 1 mug/mL, magnetically stirring for 2 hours, uniformly mixing, and adsorbing the IFN-gamma cell factor on the outer layer by utilizing aldehyde sodium alginate with negative charges on the outer layer of the GA-OSA double-layer microdroplet to obtain the GA-OSA double-layer microdroplet wrapped by the IL-4 and the IFN-gamma cell factor.
S3, dissolving type I collagen (Col-I for short) in a dilute acetic acid solution to prepare a 2wt% homogeneous collagen solution, wherein the mass ratio of collagen to nano hydroxyapatite (nHAP for short) is 1:2, uniformly dispersing 0.4g of nHAP in 10mL of collagen solution to obtain a mixed solution, pouring the mixed solution into a first space 4 of a mould (shown in figure 1), placing the mould on a device capable of forming a unidirectional temperature gradient, as shown in figure 2, placing the lower part of the mould at-196 ℃ and the upper part of the mould at room temperature, inducing separation and crystallization of different phases in the mixed solution through longitudinal temperature gradient, directionally assembling the mixed solution into an oriented lamellar collagen-nano hydroxyapatite bracket, freezing and drying after the bracket is solidified, placing the dried bracket in a closed container, and steaming and crosslinking the bracket for 12h by using 25wt% glutaraldehyde to obtain the stable oriented lamellar Col-I-nHAP bracket.
S4, placing 0.1g of the orientation plate layered Col-I-nHAP stent into 2mL of GA-OSA double-layer microdroplet liquid coated with IL-4 and IFN-gamma cytokines, soaking for 24h, depositing the GA-OSA double-layer microdroplet liquid coated with the IL-4 and IFN-gamma cytokines into the internal space structure of the orientation plate layered Col-I-nHAP stent in a liquid-liquid phase separation mode, and taking out the stent for freeze drying to obtain the bone tissue repair composite stent.
Example 2
The preparation method of the bone tissue repair composite scaffold of this example is basically the same as that of example 1, except that the concentration of the collagen solution in step S3 is 3wt%.
Example 3
The preparation method of the bone tissue repair composite scaffold of this example is basically the same as that of example 1, except that the concentration of the collagen solution in step S3 is 4wt%.
To verify whether the bone tissue repair composite scaffold of the present invention achieved the expected effect, the following performance tests were performed on the GA-OSA bilayer droplets prepared in examples 1 to 3 and the Col-I-nHAP scaffold.
1. Detection of droplet liquid structure
FIG. 3 is a microscope and a fluorescence image of a droplet, wherein A1-A2 are a microscope and a fluorescence image of a single layer droplet, respectively; B1-B2 are microscopic and fluorescent images of the bilayer droplets, respectively.
As can be seen from A1-A2 of fig. 3, the microscopic and fluorescent images of the droplet show that the droplet contains only one layer of structure, illustrating that the droplet is a single layer structure; as can be seen from B1-B2 of fig. 3, the droplet comprises a bilayer structure, illustrating that the droplet forms a bilayer structure on the basis of a monolayer droplet, indicating successful preparation of a bilayer droplet; since the cytokine is fluorescent modified, the results of the fluorescence image demonstrate that GA-OSA bilayer droplets successfully encapsulate IL-4 and IFN-gamma cytokines.
2. Col-I-nHAP scaffold assay
FIG. 4 is a scanning electron microscope image of a cross section and a longitudinal section of the Col-I-nHAP stent prepared in example 1.
FIG. 5 is a cross-sectional scanning electron microscope image of the Col-I-nHAP scaffold prepared in examples 1-3, in sequence from left to right.
From the SEM images of fig. 4 and 5, it can be seen that the scaffolds have a regular orientation structure, illustrating that the scaffolds prepared by the temperature gradient-induced method of examples 1 to 3 have an orientation structure.
3. Col-I-nHAP scaffold pore conditions
FIG. 6 is a graph of the porosity of the Col-I-nHAP scaffolds prepared in examples 1-3.
FIG. 7 is a pore distribution diagram of the Col-I-nHAP scaffold prepared in examples 1-3.
Fig. 6-7 illustrate that the scaffolds prepared in examples 1-3 have a porous structure, and scaffold materials of different porosities and pore sizes can be prepared by adjusting the composition of the raw materials. Examples 1-3 differ only in the concentration of the collagen solution, but examples 1-3 differ significantly in pore size, indicating that the effect of the concentration of the collagen solution on the pore size of the scaffold is relatively large.
What has been described above is merely some of the specific embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concept of the present invention.
Claims (7)
1. The bone tissue repair composite scaffold is characterized in that gelatin-hydroformylation sodium alginate double-layer microdroplet wrapped with IL-4 and IFN-gamma cytokines is loaded on an oriented lamellar collagen-nano hydroxyapatite scaffold, the inner layer of the gelatin-hydroformylation sodium alginate double-layer microdroplet wrapped with the IL-4 and IFN-gamma cytokines wraps the IL-4 cytokines, and the outer layer wraps the IFN-gamma cytokines;
the preparation method of the gelatin-aldehyde sodium alginate double-layer micro-droplet liquid coated with IL-4 and IFN-gamma cytokines comprises the following steps:
dissolving aldehyde sodium alginate in PBS buffer solution to obtain aldehyde sodium alginate solution, dissolving gelatin in PBS buffer solution to obtain gelatin solution, and uniformly mixing the aldehyde sodium alginate solution and the gelatin solution to obtain gelatin-aldehyde sodium alginate double-layer microdroplet with positive charges on the inner layer and negative charges on the outer layer;
adding the IL-4 cytokine into gelatin-aldehyde sodium alginate double-layer micro-droplet liquid, uniformly mixing, and adsorbing the IL-4 cytokine on the inner layer by utilizing gelatin with positive charges on the inner layer of the gelatin-aldehyde sodium alginate double-layer micro-droplet liquid to obtain gelatin-aldehyde sodium alginate double-layer micro-droplet liquid coated with the IL-4 cytokine;
adding IFN-gamma cytokines into gelatin-hydroformylation sodium alginate double-layer microdroplet wrapped with IL-4 cytokines, uniformly mixing, and adsorbing the IFN-gamma cytokines on the outer layer by using aldehyde sodium alginate with negative charges on the outer layer of the gelatin-hydroformylation sodium alginate double-layer microdroplet to obtain gelatin-hydroformylation sodium alginate double-layer microdroplet wrapped with IL-4 and IFN-gamma cytokines;
the preparation method of the oriented plate lamellar collagen-nano hydroxyapatite scaffold comprises the following steps:
dissolving type I collagen in a solvent to prepare a collagen solution, uniformly dispersing nano hydroxyapatite in the collagen solution to obtain a mixed solution, then utilizing unidirectional temperature gradient to induce the mixed solution to directionally assemble an oriented lamellar collagen-nano hydroxyapatite scaffold, freeze-drying the scaffold after solidification, and utilizing glutaraldehyde to steam and crosslink the scaffold to obtain a stable oriented lamellar collagen-nano hydroxyapatite scaffold; the solvent is acetic acid or hydrochloric acid solution, the concentration of type I collagen in the collagen solution is 2-4wt%, and the mass ratio of the collagen to the nano hydroxyapatite is 1:2; the unidirectional temperature gradient is-196 ℃ to room temperature.
2. The bone tissue repair composite scaffold according to claim 1, wherein the preparation method of the aldehyde sodium alginate comprises the following steps:
according to parts by weight, 5-10 parts of sodium alginate are dissolved in 200 parts of water, 50-100 parts of absolute ethyl alcohol is added after stirring uniformly, 5-10 parts of sodium periodate is added after stirring uniformly and is reacted for 12-24 hours at room temperature in a dark place, then 10-20 parts of ethylene glycol is added to neutralize excessive sodium periodate, the reaction is continued for 2-4 hours in a dark place, 5-10 parts of sodium chloride is added after the reaction is finished, 600-1200 parts of ethanol is used for precipitating out oxidized sodium alginate, then the obtained precipitate is added into water for dissolution to obtain a solution, then ethanol is added for precipitating out oxidized sodium alginate in the solution, the dissolving-precipitating process is repeated for 2-5 times to obtain an aldehyde sodium alginate solution, and finally the aldehyde sodium alginate solution is purified and freeze-dried to obtain the aldehyde sodium alginate.
3. The bone tissue repair composite scaffold according to claim 1, wherein the concentration of the aldehyde sodium alginate solution is 2-5wt%, the concentration of the gelatin solution is 2-10wt%, and the volume ratio of the aldehyde sodium alginate solution to the gelatin solution is 1:1.
4. The bone tissue repair composite scaffold of claim 1, wherein the concentration of IL-4 cytokine is 1 μg/mL, the concentration of IFN- γ cytokine is 1 μg/mL, and the electrostatic interaction time of IL-4 cytokine and IFN- γ cytokine with gelatin-formylated sodium alginate bilayer droplets is 2h.
5. The method for preparing the bone tissue repair composite scaffold according to any one of claims 1 to 4, which is characterized by comprising the following steps:
soaking the oriented lamellar collagen-nano hydroxyapatite scaffold in gelatin-aldehyde sodium alginate double-layer micro-droplet liquid coated with IL-4 and IFN-gamma cytokines for 12-24 hours, and then taking out the scaffold for freeze drying to obtain the bone tissue repair composite scaffold.
6. The method for preparing a composite scaffold for repairing bone tissue according to claim 5, wherein the mass-volume ratio of the oriented plate lamellar collagen-nano hydroxyapatite scaffold to the gelatin-hydroformylation sodium alginate bilayer microdroplet coated with IL-4 and IFN-gamma cytokines is 0.05-0.2g/mL.
7. The use of the bone tissue repair composite scaffold according to any one of claims 1 to 4 in the preparation of tissue engineering materials or bone defect repair materials.
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| CN114949371A (en) * | 2022-04-12 | 2022-08-30 | 重庆大学 | Double-layer porous scaffold for repairing articular cartilage defect and preparation method thereof |
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| CN112370572A (en) * | 2020-11-23 | 2021-02-19 | 中国人民解放军陆军军医大学第一附属医院 | Bone repair material for treating large bone defect and preparation method and application thereof |
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