CN114940967A - Breast milk extracellular vesicle and application thereof in preparation of bone repair material - Google Patents
Breast milk extracellular vesicle and application thereof in preparation of bone repair material Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0631—Mammary 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
- A61L27/36—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
- A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial 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
- A61L27/52—Hydrogels or hydrocolloids
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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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- 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
- 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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- C—CHEMISTRY; METALLURGY
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention discloses a breast milk extracellular vesicle and application thereof in preparing bone repair materials, wherein the preparation of the breast milk extracellular vesicle comprises the following steps: collecting fresh human breast milk, centrifuging to remove fat, and taking supernatant; then centrifuging to remove cells and debris, and taking the supernatant; filtering with a filter membrane, centrifuging the obtained supernatant, collecting precipitate, and adding a solvent to resuspend the precipitate; centrifuging again to remove the solvent, and adding a small amount of solvent to resuspend the precipitate; finally, filtering the mixture by using a filter membrane to obtain the breast milk extracellular vesicles. The breast milk extracellular vesicles can be taken by mesenchymal bone marrow stem cells, so that the transcription levels of the mesenchymal bone marrow stem cells Runx2, Alp and Bmp2 protein are improved, and the mesenchymal bone marrow stem cells are promoted to be differentiated into osteoblasts. The breast milk extracellular vesicles are loaded on a bone tissue engineering scaffold material, so that bone regeneration can be remarkably promoted.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to breast milk extracellular vesicles and application thereof in preparation of bone repair materials.
Background
Bone grafting is one of the most commonly used surgical methods in bone surgery to promote bone regeneration. In all clinically available bone grafts, autologous bone is still considered the gold standard because of all the necessary properties required for bone regeneration, osteoconduction, osteoinduction and osteogenesis are combined. However, concerns of limited supply and donor complications still exist. Because the allogenic bone graft has various forms and a large number, the allogenic bone graft is dominant in the orthopedic bone graft. However, allograft bone transplantation results in poor healing compared to the use of autograft, and there is also the potential for disease transmission and the transmission of other infectious agents. More importantly, the number of natural bone grafts available traditionally remains far from meeting clinical needs.
To address these limitations, synthetic bone substitutes have become one of the most potential markets for the orthopedic industry. Among them, calcium phosphate (Cap) -based biomaterials (such as hydroxyapatite (Hap), Cap cement and ceramics) and recombinant human bone morphogenetic proteins (rhBMP, such as rhBMP-2 and rhBMP-7) are most widely used, either alone or in combination. The original bone substitute generally only has bone conductivity and is mainly applied to reconstruction of large bone defects, and the recombinant human bone morphogenetic protein basically has bone induction effect and has the capability of promoting fracture healing. However, recombinant human bone morphogenic proteins have received much attention in clinical applications due to their supraphysiological dose, poor clinical outcome and cost issues. Therefore, research and development of more drugs or biomaterials are urgently needed, and more effective ways are provided for promoting bone regeneration.
Disclosure of Invention
The invention aims to provide breast milk extracellular vesicles and application thereof in preparing bone repair materials.
The purpose of the invention is realized by the following technical scheme:
a preparation method of breast milk extracellular vesicles comprises the following steps:
collecting human fresh breast milk, centrifuging at a centrifugal force of 500-5000 Xg (preferably 2000 Xg), centrifuging for 5-30 min to remove fat, and taking supernatant; 10000-16000 Xg (preferably 12000 Xg), centrifuging for 15-45 min (preferably 30min) to remove cells and debris, and taking the supernatant; filtering with a filter membrane, centrifuging the obtained supernatant 100000-160000 Xg (preferably 12000 Xg) for 2-24 h (preferably 2h), collecting the precipitate, and adding a solvent to resuspend the precipitate; then, carrying out superseparation for 2-24 h (preferably 2h) by 100000-160000 Xg (preferably 12000 Xg), removing the solvent, and adding a small amount of solvent to resuspend the precipitate; finally, filtering the mixture by using a filter membrane to obtain breast milk extracellular vesicles;
the operations are all carried out in an environment of 4 ℃;
the solvent is a solvent which has biocompatibility with breast milk extracellular vesicles and is a buffer solution or a liquid culture medium;
the buffer solution is preferably PBS buffer solution, the pH value is 6.5-7.5, and the pH value is preferably 7;
the liquid culture medium is one or more selected from DMEM liquid culture medium, MEM liquid culture medium, RPMI 1640 liquid culture medium, F12 liquid culture medium or IMDM liquid culture medium;
the filtration membrane (filtration head of the needle sterilization filter) is a 0.1 to 1 μm micro-porous sterilization filtration membrane, preferably a 0.22 μm micro-porous sterilization filtration membrane.
The breast milk extracellular vesicle prepared by the method has the size of 30-1000 nm, the protein concentration of 0.01-1000 mu g/mL and the particle number of 1 multiplied by 10 8 ～1×10 13 /mL；
When the size is too small (less than 30nm), the existing centrifugation conditions cannot be met; when oversized (greater than 1000nm), the extracted may be cellular debris, rather than extracellular vesicles;
preferably, the protein concentration is 0.1-100 mug/mL; more preferably, the protein concentration is 1-50 mug/mL; even more preferably, the protein concentration is 25 μ g/mL;
preferably, the number of particles is 1X 10 8 ～1×10 13 Per mL; more preferably, the number of particles is 1X 10 9 ～1×10 12 Per mL; even more preferably, the number of particles is 3X 10 11 /mL。
The application of the breast milk extracellular vesicles in preparing bone repair materials; the breast milk extracellular vesicles can be taken up by mesenchymal bone marrow stem cells, and Akt phosphorylation and ERK (1/2) phosphorylation are activated at 6h and 12h respectively; the breast milk extracellular vesicles can continuously improve the transcription level of Runx2, Alp and Bmp2 proteins of mesenchymal bone marrow stem cells on days 3, 5, 7 and 12 through gene regulation, and promote the differentiation of the mesenchymal bone marrow stem cells to osteoblasts.
A bone repair material is characterized in that breast milk extracellular vesicles are adsorbed in a bone tissue engineering scaffold material, wherein the volume ratio of the bone tissue engineering scaffold material to the breast milk extracellular vesicles is 1: 0.5-2.
The bone tissue engineering scaffold material is freeze-dried hydrogel, medical sponge or medical foam;
the hydrogel may be a hydrogel formed by physical action, such as a gel formed by blending polyacrylamide with polyacrylic acid, or the like; gel formed by interaction of water-soluble polymer and metal ion, such as sodium alginate and chitosan, which respectively form sodium alginate, chitosan hydrogel and the like with calcium ion and phosphate radical; hydrogel formed by interaction of polycation electrolyte and polyanion electrolyte, such as alginic acid-chitosan hydrogel formed by interaction of alginic acid and chitosan; high molecular chain polymerization to form hydrogel, such as gelatin and agar;
preferably, the hydrogel is double-bond modified hyaluronic acid;
the double-bond modified hyaluronic acid is prepared by the following steps:
reacting hyaluronic acid with methacrylic anhydride or acryloyl chloride to obtain double-bond modified hyaluronic acid capable of being photo-crosslinked; dissolving the double-bond modified hyaluronic acid, adding a photoinitiator, removing bubbles, placing the mixture in a mould, and initiating polymerization reaction under the irradiation of ultraviolet light to obtain the double-bond modified hyaluronic acid.
Compared with the prior art, the invention has the following advantages and effects:
the breast milk extracellular vesicles of the present invention carry abundant bioactive substances such as non-coding molecules of RNA, mRNA, DNA, protein, etc., and effectively mediate intercellular and organ-to-organ communication by delivering specific cargo. Research shows that the breast milk extracellular vesicles can be taken up by mesenchymal bone marrow stem cells, Akt phosphorylation and ERK (1/2) phosphorylation are activated at 6h and 12h respectively, and transcription levels of Runx2, Alp and Bmp2 proteins of the mesenchymal bone marrow stem cells can be continuously improved on days 3, 5, 7 and 12 through gene regulation, so that the mesenchymal bone marrow stem cells are promoted to be differentiated into osteoblasts. The breast milk extracellular vesicles are loaded on a bone tissue engineering scaffold material and used for a rat skull defect model, and the bone tissue engineering scaffold based on the breast milk extracellular vesicles is found to be capable of remarkably promoting bone regeneration.
In addition, the material based on the breast milk extracellular vesicle bone tissue engineering scaffold is derived from food breast milk, and the preparation process is simple, green and safe and is easy to realize; therefore, the bone tissue engineering scaffold provided by the invention has wide popularization and application prospects in serving as or preparing products or products for promoting bone defect regeneration formation.
Drawings
FIG. 1 is a transmission electron micrograph of breast milk extracellular vesicles.
Fig. 2 is a nanoflow result graph of breast milk extracellular vesicles.
FIG. 3 is a diagram showing the result of Western blotting to determine whether the extracted breast milk extracellular vesicles contain CD9 and CD63 proteins.
FIG. 4 shows the activation of Akt phosphorylation by different concentrations of extracellular vesicles and decellularized extracellular vesicle breast milk.
FIG. 5 shows the activation of ERK (1/2) phosphorylation by breast milk with different concentrations of extracellular vesicles and decellularized vesicles.
FIG. 6 shows the effect of different concentrations of extracellular vesicles of human milk and the extracellular vesicle-removed human milk on the transcription level of the osteogenic factor Runx2 after rat bone marrow mesenchymal stem cells are treated for 3, 5, 7 and 12 days.
FIG. 7 shows the effect of different concentrations of extracellular vesicles of human milk and the extracellular vesicle-removed human milk on the transcription level of osteogenic factor Alp after rat bone marrow mesenchymal stem cells were treated for 3, 5, 7 and 12 days, respectively.
FIG. 8 shows the effect of different concentrations of extracellular vesicles of human milk and the extracellular vesicle-removed human milk on the transcription level of the osteogenic factor Bmp2 after rat bone marrow mesenchymal stem cells are treated for 3, 5, 7 and 12 days respectively.
FIG. 9 shows the effect of breast milk extracellular vesicles on bone regeneration after skull defect in rats.
FIG. 10 is a graph showing the results of HE staining and Masson staining of breast milk extracellular vesicles after 1 month of repair of rat cranial defects.
FIG. 11 is a graph showing the results of HE staining and Masson staining of breast milk extracellular vesicles and breast milk with extracellular vesicles after 3 months of repair of rat skull defects.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Example 1
The preparation of the breast milk extracellular vesicles and the bone tissue engineering scaffold comprises the following steps:
s1, collecting fresh human breast milk, and centrifuging at a rotating speed of 2000 Xg for 10min to remove fat in an environment at 4 ℃ by adopting a differential centrifugation method; then 12000 Xg, centrifugate for 30min to remove cells and debris; then filtering the obtained supernatant by using a 0.22 mu m filter head; finally, placing the obtained supernatant in a super-speed centrifuge with the volume of 120,000 Xg for super separation for 2 h; collecting the supernatant (breast milk solution for removing extracellular vesicles), and resuspending the extracellular vesicle pellet with a large amount of PBS buffer (pH 7, the same below); then 120,000 Xg, super-separating for 2h, removing the PBS buffer solution, and adding a small amount of PBS buffer solution to resuspend extracellular vesicle precipitates; resuspending overnight at 4 ℃, and filtering by using a 0.22 mu m filter head to obtain breast milk extracellular vesicles;
s2, detecting the protein content of the breast milk extracellular vesicles obtained in the step S1 by a BCA protein quantitative method, diluting the protein content to 25 mu g/mL by PBS, and adsorbing the diluted protein content to a freeze-dried methacrylic anhydridized hyaluronic acid hydrogel 3D scaffold (namely double-bond modified hyaluronic acid hydrogel with the diameter of 5mm and the height of 2mm), wherein the volume ratio of the 3D scaffold to the outer vesicles is 1:1, so that the bone tissue engineering scaffold can be obtained.
Example 2
The preparation of the breast milk extracellular vesicles and the bone tissue engineering scaffold comprises the following steps:
s1, the same as the embodiment 1;
s2, diluting the breast milk extracellular vesicles obtained in the step S1 (5 mug/mL), and adsorbing the diluted cells into a sponge, wherein the volume ratio of the sponge to the extracellular vesicles is 1:1/2, so that the bone tissue engineering scaffold can be obtained.
Example 3
The preparation of the breast milk extracellular vesicles and the bone tissue engineering scaffold comprises the following steps:
s1, the same as the embodiment 1;
s2, diluting the breast milk extracellular vesicles obtained in the step S1 (50 mu g/mL), and adsorbing the diluted cells into foams (diameter is 5mm, height is 2mm), wherein the volume ratio of the foams to the extracellular vesicles is 1:1, so that the bone tissue engineering scaffold can be obtained.
Example 4
The preparation of the breast milk extracellular vesicles and the bone tissue engineering scaffold comprises the following steps:
s1, the same as the embodiment 1;
s2, diluting the breast milk extracellular vesicles obtained in the step S1 (500 mu g/mL), and adsorbing the diluted cells into a sponge (5 mm in diameter and 2mm in height), wherein the volume ratio of the sponge to the extracellular vesicles is 1:2/3, so that the bone tissue engineering scaffold can be obtained.
Example 5
The preparation of the breast milk extracellular vesicles and the bone tissue engineering scaffold comprises the following steps:
s1, the same as the embodiment 1;
s2, diluting the breast milk extracellular vesicles obtained in the step S1 (1000 mug/mL), and adsorbing the diluted cells into foams (diameter is 5mm, height is 2mm), wherein the volume ratio of the foams to the extracellular vesicles is 1:2, so that the bone tissue engineering scaffold can be obtained.
Effect example 1
Appearance of breast milk extracellular vesicles observed by TEM
1. Experimental methods
(a) Diluting the breast milk extracellular vesicles of the example to 10 μ g/mL with PBS buffer, and mixing well;
(b) taking out the TEM copper mesh by using a pair of tweezers, and placing the TEM copper mesh under a heat lamp;
(c) sucking diluted breast milk extracellular vesicles by using a 10-mu-L gun head, dripping about 5-mu-L breast milk extracellular vesicles on a copper mesh, turning on a heat lamp, heating for about 20 minutes, and waiting for the copper mesh to be basically dry;
(d) and taking out the copper mesh, placing the copper mesh in the air, air-drying for 30 minutes, and placing the copper mesh in a biological transmission electron microscope to observe the appearance of the breast milk extracellular vesicles.
2. Results of the experiment
The appearance of the breast milk extracellular vesicles observed by TEM is shown in fig. 1, and it can be seen that the breast milk extracellular vesicles have a structure of a lipid bilayer membrane in a shape of a cup.
Effect example 2
Nano-flow meter for detecting quantity and size of breast milk extracellular vesicles
1. Experimental methods
The number and size of the breast milk extracellular vesicles obtained in step S1 of example were measured by a nanoflow meter.
2. Results of the experiment
The number and size of the breast milk extracellular vesicles are shown in FIG. 2, the size of the extracted breast milk extracellular vesicles is 40-150 nm, the average size is 66.75nm, and each milliliter of breast milk solution contains 2.99 × 10 11 An extracellular vesicle.
Effect example 3
Western blotting characterization of the Breast milk extracellular vesicle surface marker proteins CD9 and CD63
1. Experimental methods
(a) Adding a loading buffer (biosharp, BL502B) to the breast milk extracellular vesicle solution prepared in the example step S1, and adding a loading buffer to the extracellular vesicle-removed breast milk solution (prepared in the example 1 step S1) of the same protein concentration;
(b) boiling the above sample for 5min, and mixing;
(c) adding 20 mu L of breast milk extracellular vesicle loading buffer and breast milk solution loading buffer for removing extracellular vesicles into 10 comb concentrated gel of an electrophoresis tank, wherein the volume of the breast milk solution loading buffer is 70V, 1h, 100V and 1.5 h;
(d) taking out the separation gel from the electrophoresis tank, preparing for membrane transfer, and carrying out treatment at 200mA for 1.5 h;
(e) taking out the transferred PVDF membrane, cutting off a band with a blade corresponding to the molecular weight of the protein, washing for 3 times by using a TBST solution, and sealing for 2 hours at room temperature by using 5% skimmed milk;
(f) the strips were then placed in cassettes containing CD9 and CD63 primary antibodies, respectively, and incubated overnight at 4 ℃;
(g) washing with TBST solution for 3 times, adding enzyme-labeled secondary antibody into the box, and incubating at room temperature for 2 h;
(h) the TBST solution was washed 3 times and finally imaged with a chemiluminescence instrument.
2. Results of the experiment
As shown in FIG. 3, the results of Western blotting detection of the surface marker protein of the breast milk extracellular vesicles are shown, wherein the breast milk extracellular vesicle group (HBM-Exos) positively expresses CD9 and CD63, and the breast milk solution (Exos-depleted HBM) from which the extracellular vesicles are removed negatively expresses CD9 and CD 63. The above results indicate that the extracted are indeed extracellular vesicles.
Effect example 4
Western blotting to test the influence of breast milk extracellular vesicles on phosphorylation of BMSC Akt and ERK (1/2)
1. Experimental methods
(a) Treating BMSCs from rats with whole culture medium (MEM-alpha medium and 10% FBS) containing breast milk extracellular vesicles and a solution of breast milk without extracellular vesicles (as a control group) at a final protein concentration of 25. mu.g/mL, adding cell lysate (biosharp, RIPA-BL504A) at different time points to collect cell proteins, centrifuging to measure the protein concentration, and adding a loading buffer;
(b) boiling the above sample for 5min, and mixing;
(c) respectively adding 20 mul of protein samples into 10 comb concentrated gel of an electrophoresis tank, wherein the protein samples are 70V, 1h, 100V and 1.5 h;
(d) taking out the separation gel from the electrophoresis tank, preparing for membrane transfer, and carrying out treatment at 200mA for 1.5 h;
(e) taking out the transferred PVDF membrane, cutting off a band with a blade corresponding to the molecular weight of the protein, washing for 3 times by using a TBST solution, and sealing for 2 hours at room temperature by using 5% skimmed milk;
(f) the bands were then placed in cassettes containing Akt, p-Akt, ERK (1/2), p-ERK (1/2), β -actin and β -tubulin primary antibodies, respectively, and incubated overnight at 4 ℃;
(g) washing with TBST solution for 3 times, adding enzyme-labeled secondary antibody into the box, and incubating at room temperature for 2 h;
(h) the TBST solution was washed 3 times and finally imaged with a chemiluminescence instrument.
2. Results of the experiment
The effect of human milk extracellular vesicles on BMSC Akt and ERK (1/2) phosphorylation results are shown in fig. 4 and 5, where Akt and ERK (1/2) phosphorylation were activated in BMSC 6h and 12h treated with whole medium containing human milk extracellular vesicles compared to Control (Control), but Akt and ERK (1/2) phosphorylation were not activated by whole medium treatment of BMSC with breast milk solution without extracellular vesicles. The above results indicate that breast milk extracellular vesicles can activate Akt and ERK (1/2), contributing to BMSC osteogenic differentiation.
Effect example 5
Test of influence of breast milk extracellular vesicles on transcription levels of osteogenic factors Runx2, Alp and Bmp2 genes
1. Experimental methods
(a) BMSCs are inoculated in a 6-well plate, and a total culture medium (MEM-alpha culture medium and 10% FBS) is added for adherence;
(b) removing old medium, adding bone induction medium (MEM-alpha medium, 10% FBS, 10mM beta-glycerophosphate, 50 mu M ascorbic acid and 10nM dexamethasone) into the control group, and respectively adding bone induction medium containing extracellular vesicles of breast milk with protein concentration of 5 mu g/mL and 25 mu g/mL or extracellular vesicle-removed breast milk solution with protein concentration of 5 mu g/mL and 25 mu g/mL into the experimental group;
(c) after 3, 5, 7 and 12 days of incubation, removing the culture medium, washing the cells for 2 times with PBS, adding 1mL of Trizol solution into each sample, uniformly blowing, and standing in a 1.5mL EP tube at room temperature for 10 min;
(d) adding 200 μ L chloroform into each tube, shaking vigorously for 15s, standing at room temperature for 5min, 4 deg.C, 14000g, and centrifuging for 15 min; taking 200-400 mu L of supernatant in a new EP tube, adding 0.5mL of isopropanol into each tube, standing at room temperature for 10min, keeping the temperature at 4 ℃, keeping the temperature at 14000g, and centrifuging for 15 min; discarding the supernatant, adding ice for precooling, washing with 75% ethanol (DEPC water) for 2 times, 12000g, and centrifuging for 5 min; discarding the supernatant, naturally drying at room temperature for 5-10 min, and dissolving RNA by using 20 mu LDEPC water;
(e) detecting the purity and concentration of RNA by using a nucleic acid protein detector;
(f) taking 1 mu g of RNA per 20 mu L of system for reverse transcription;
(g) the qRT-PCR reaction was performed by using a PCR instrument, and the transcription levels of the osteogenic factor Runx2, Alp and Bmp2 genes were detected.
2. Results of the experiment
Effects of breast milk extracellular vesicles on transcription levels of osteogenic factors Runx2, Alp and Bmp2 as shown in fig. 6 to 8, breast milk extracellular vesicles (5HBM-Exos +) having a protein content of 5 μ g/mL can promote transcription levels of Runx2 and Alp on day 3, and as the content of breast milk extracellular vesicles is increased (25 HBM-Exos +), transcription levels of Runx2 and Alp are increased, but breast milk from which extracellular vesicles are removed (5 Exos-purified HBM + and 25 Exos-purified HBM +) does not show a promoting effect, compared to a control group; after 5, 7 and 12 days of treatment, the 25HBM-Exos group can increase the transcription level of Runx2, Alp and Bmp2 genes all the time, however, the 5 Exos-deleted HBM + and 25 Exos-deleted HBM + groups show up-regulation of Runx2, Alp and Bmp2 gene transcription only at 12 days, but their promotion effect is still weaker than that of the 25HBM-Exos + group. The results show that the breast milk extracellular vesicles can effectively promote the transcription levels of BMSC osteogenesis factors Runx2, Alp and Bmp2 genes, and show good osteogenesis promoting capability.
Effect example 6
Influence of bone tissue engineering scaffold based on breast milk extracellular vesicles on bone defect regeneration
1. Experimental methods
(1) Skull defect model
(a) Selecting male SD rats with the weight of 230-260 g as study objects;
(b) injecting barbital sodium into abdominal cavity of all rats for anesthesia, shaving skull, cutting skin of skull with a blade, taking off bone with a rotary head with diameter of 5mm, and making bone defect model;
(2) preparation of bone tissue engineering scaffold based on breast milk extracellular vesicles
Same as step S2 of example 1;
(3) medicine for bone defect position
The bone defect part of the control group is filled with methacrylic acid anhydridized hyaluronic acid 3D scaffold (HA-MA) soaked by PBS buffer solution, the bone defect part of the experimental group is filled with grade-A crylic acid anhydridized hyaluronic acid 3D scaffold adsorbed with breast milk extracellular vesicles or extracellular vesicle-removed breast milk solution (Exos-depleted HBM/HA-MA), and sampling is respectively carried out at 1 month, 2 months and 3 months to observe the bone regeneration condition.
2. Results of the experiment
The micro-CT, HE staining and Masson staining results of the bone tissue engineering scaffold based on the breast milk extracellular vesicles, which is prepared by the invention, on the influence of the bone defect regeneration are shown in figures 9, 10 and 11. FIG. 9 shows that breast milk extracellular vesicle-based scaffolds for bone tissue engineering (HBM-Exos/HA-MA) are most able to promote bone regeneration in bone defect repair for either 1 month, 2 months or 3 months. And the analysis of trabecular bone thickness and trabecular bone number can also obtain consistent results through the ratio of the volume of the regenerated bone to the total volume; histological analysis (HE and Masson staining) was performed on 1 month skull specimens, and it can be seen from FIG. 10 that more trabecular regeneration and more collagen precipitate formation were seen in the HBM-Exos/HA-MA group; the 3 month skull sample shown in fig. 11 also demonstrates that the bone tissue engineering scaffold containing breast milk extracellular vesicles is more conducive to bone regeneration.
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 modifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of breast milk extracellular vesicles is characterized by comprising the following steps:
collecting human fresh breast milk, centrifuging at a centrifugal force of 500-5000 Xg for 5-30 min to remove fat, and taking supernatant; then 10000-16000 Xg, centrifuging for 15-45 min to remove cells and debris, and taking the supernatant; filtering by using a filter membrane, centrifuging the obtained supernatant 100000-160000 Xg for 2-24 h, collecting the precipitate, and adding a solvent to resuspend the precipitate; then, carrying out super separation for 2-24 h by 100000-160000 Xg, removing the solvent, and adding a small amount of solvent to carry out heavy suspension precipitation; finally, filtering the mixture by using a filter membrane to obtain the breast milk extracellular vesicles.
2. The method of claim 1, wherein: the solvent is buffer solution or liquid culture medium.
3. The method of claim 2, wherein:
the buffer solution is PBS buffer solution, and the pH value is 6.5-7.5;
the liquid culture medium is one or more of DMEM liquid culture medium, MEM liquid culture medium, RPMI 1640 liquid culture medium, F12 liquid culture medium or IMDM liquid culture medium.
4. The method of claim 1, wherein:
the filtering membrane is a 0.1-1 mu m micropore degerming filtering membrane;
the operation of the method is carried out in an environment of 4 ℃.
5. A human milk extracellular vesicle produced by the method according to any one of claims 1 to 4.
6. The breast milk extracellular vesicle of claim 5, wherein: the size is 30-1000 nm, the protein concentration is 0.01-1000 mug/mL, and the particle number is 1 multiplied by 10 8 ～1×10 13 /mL。
7. Use of the breast milk extracellular vesicles of claim 5 or 6 in the preparation of a bone repair material.
8. A bone repair material characterized by: the breast milk extracellular vesicles according to claim 5 or 6 are adsorbed to a scaffold material for bone tissue engineering.
9. Bone repair material according to claim 8, characterized in that: the bone tissue engineering scaffold material is freeze-dried hydrogel, medical sponge or medical foam.
10. The bone repair material of claim 9, wherein: the hydrogel is double-bond modified hyaluronic acid.
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| CN202210496280.4A CN114940967A (en) | 2022-05-09 | 2022-05-09 | Breast milk extracellular vesicle and application thereof in preparation of bone repair material |
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