CN116350676B - Medicine for promoting periodontal bone reconstruction by stem cell exosome - Google Patents
Medicine for promoting periodontal bone reconstruction by stem cell exosome Download PDFInfo
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- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/488—Pueraria (kudzu)
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/534—Mentha (mint)
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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Abstract
The application relates to the technical field of high-efficiency osteogenesis, in particular to a medicament for promoting periodontal bone reconstruction by utilizing stem cell exosomes, which can effectively enhance the bone differentiation of periodontal bone cells by adding the odontogenic exosomes on the basis of original grinding medicaments, and in addition, the extract of kudzuvine root and the extract of peppermint are screened from a plurality of reported traditional Chinese medicines with osteogenesis differentiation, and according to the research of clinical cases, the extract can effectively promote the regrowth of cementum wires, increase bone level and recover bone trabeculae, and can increase the bone level by more than 4.0mm after reasonable proportion of the two inducers, compared with the prior art, the medicament has better effect in 3.0mm improvement, and better effect in periodontal bone reconstruction medicaments.
Description
[ field of technology ]
The application relates to the technical field of efficient bone formation, in particular to a medicament for promoting periodontal bone reconstruction by utilizing stem cell exosomes.
[ background Art ]
Periodontitis is a chronic inflammation that invades the gums and periodontal tissues, a destructive disease characterized mainly by the formation of periodontal pockets and inflammation of the pocket walls, periodontal bone loss, a group of characteristics of dental loosening, malignant diseases of the oral cavity, applicant's prior application patent: 202110311551.X proposes the use of mesenchymal stem cell gel, allogeneic bone powder and inducer for the preparation of a medicament for the reconstruction of periodontal bone, however, the specific inducer composition is not clear.
In intensive researches of the applicant, it is found that although mesenchymal stem cell gel can promote bone differentiation of cells, impurities in the mesenchymal stem cell gel are still more, bone differentiation effect is not optimal, peripheral bone heightening effect is not optimal, certain traditional Chinese medicines reported in the prior art have the effects of tonifying kidney and strengthening yang, strengthening tendons and bones, dispelling wind and removing dampness, pharmacological and clinical researches prove that the mesenchymal stem cell gel has good effects of preventing and treating osteoporosis, can promote osteoblast proliferation and mineralization and inhibit osteoclast activity, and the traditional Chinese medicine components can be used as inducers of medicines, however, in specific researches, it is found that part of the traditional Chinese medicine preparations have obvious inhibition effect on cell proliferation, inhibition effect even leads to bone cell growth not to be good, and in addition, inducer with the effect of promoting bone differentiation is reported to have promotion effect, some have even inhibition effect in periodontal bone reconstruction process, so that the screening of the inducer is particularly important in the medicines.
[ application ]
In view of the foregoing, there is a need for screening and research on inducers that are effective in promoting the application of mesenchymal stem cells in the preparation of periodontal bone reconstruction drugs.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
a medicament for promoting periodontal bone reconstruction by utilizing stem cell exosomes, which comprises the following components in percentage by mass: 15% of mesenchymal stem cell gel, 4% of odontogenic stem cell exosomes, 80% of bone meal and 1% of inducer; the inducer is radix Puerariae extract and/or herba Menthae extract.
Furthermore, the kudzuvine root extract and the mint extract are ethanol extracts.
Further, the ethanol of the ethanol extract is 70% ethanol solution by volume.
Further, the mass ratio of the kudzuvine root extract to the peppermint extract is 1:3.
Further, the specific extraction method of the kudzuvine root extract and the peppermint extract comprises the following steps: 500g of dry kudzuvine root, dry epimedium herb, fresh peppermint and fresh lotus leaf medicinal materials are respectively weighed, and are refluxed for 2 times with ethanol solution with the volume percentage of 70 percent for 2 hours each time, then the extract is concentrated to 20g, and the corresponding extract is obtained after storage at the temperature of 4 ℃.
Further, the odontogenic stem cell exosome markers are TSG101, CD81 and CD63 proteins.
Further, the preparation method of the tooth source stem cell exosome comprises the following steps:
(1) Inoculating dental pulp stem cells into a culture dish or a culture bottle, discarding old culture medium when the growth of the cells is fused to 80%, washing 3 times by PBS, and adding a basal culture medium;
(2) After culturing for 24-48 hours, collecting the cell culture solution;
(3) DMEM washed with PBS to remove dead cells and replaced with 10% fbs;
(4) Continuously collecting about 500ml of cell culture solution, and sub-packaging into 50ml centrifuge tubes;
centrifuging to remove macromolecular vesicles, concentrating in a 100KD ultrafiltration tube, discarding liquid with a height of 1.5cm from the bottom of the centrifugation tube, and centrifuging at 5000g at 4deg.C for 10min to obtain culture medium concentrate; 5ml of sucrose solution with the volume percentage of 30% is injected into the concentrated culture medium solution, and after layering occurs, the concentrated culture solution is continuously added, so that the liquid level is up to the position of a pipe orifice of 3 mm; centrifuging, removing the upper culture medium, and gently sucking sucrose solution containing exosomes below by using a syringe; and (3) after collecting sucrose solution, diluting with PBS buffer solution, placing into an ultrafiltration tube with 100KD, balancing, repeatedly washing with PBS, centrifuging until removing sucrose heavy water solution, and finally collecting concentrated solution which is an exosome of dental stem cells, filtering, sterilizing, and storing in a refrigerator at-80 ℃.
The application has the following beneficial effects:
according to the technical scheme, the odontogenic exosome can effectively enhance the bone differentiation of periodontal bone cells, in addition, the pueraria extract and the peppermint extract are screened from a plurality of reported traditional Chinese medicines with the bone differentiation through screening of inducers, and according to the research of clinical cases, the extract can effectively promote the regrowth of cementum wires, increase the bone level and restore bone trabeculae, and after the reasonable proportion of the two inducers, the bone level can be increased to more than 4.0mm, and compared with the prior art, the 3.0mm improvement effect is better, and the effect is better in periodontal bone reconstruction medicines.
[ description of the drawings ]
FIG. 1 is a morphology of a mesenchymal stem cell gel;
FIG. 2 is a morphology of exosomes under electron microscopy;
FIG. 3 is a graph showing the analysis of NTA particle size of exosomes;
FIG. 4 is a graph of Western Blot detection results of exosomes;
fig. 5-12 are x-ray images taken before and after treatment of a partial case.
[ detailed description ] of the application
The following detailed description of the present application will provide further details in order to make the above-mentioned objects, features and advantages of the present application more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. The application may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the application, which is therefore not limited to the specific embodiments disclosed below.
Example 1:
1. mesenchymal stem cell gel acquisition:
and collecting 10mL of venous peripheral blood, namely putting the peripheral blood into a variable speed centrifuge for centrifugation for three minutes, after the variable speed centrifugation is finished, beating a blood vessel in a sterile operation table, taking out jelly-like PRF, transferring to a disinfection vessel, shearing to remove upper-layer pale yellow colloid, and simply cleaning erythrocytes in the jelly with 5mL of physiological saline to retain the colloid, thereby obtaining mesenchymal stem cell gel, wherein the colloid shape is shown in figure 1.
And (3) carrying out culture increase and substitution transfer by using a special culture medium (purchased from HYCLONE company) for mesenchymal stem cells, and harvesting p 4-generation cells.
After that, immunofluorescent staining was performed, and the mesenchymal stem cell factor protein markers CD73, CD90 and CD105 were positive according to the detection standard of the International cell therapy Union (see, in particular, patent document: 202110311551. X).
2. Obtaining tooth source stem cell exosomes:
obtaining odontogenic stem cells from deciduous teeth and adult corrective tooth extraction of normal infants; the missing teeth are from the normal tooth extraction of the south peace paste, wound and beauty oral outpatient department.
1. Acquisition of odontogenic stem cell exosomes:
(1) Mixing the deciduous dental pulp stem cells of the young children and the adult dental pulp stem cells, inoculating the mixed deciduous dental pulp stem cells into a culture dish or a culture bottle, discarding old culture medium when the growth of the cells is fused to 80%, washing for 3 times by PBS, and adding a basic culture medium;
(2) After culturing for 24-48 hours, collecting the cell culture solution;
(3) DMEM washed with PBS to remove dead cells and replaced with 10% fbs;
(4) Continuously collecting about 500ml of cell culture solution, and sub-packaging into 50ml centrifuge tubes;
(5) Centrifuging to remove macromolecular vesicles, concentrating in a 100KD ultrafilter tube, discarding liquid with a height of 1.5cm from the bottom of the tube, centrifuging at 5000g at 4deg.C for 10min to obtain culture medium concentrate; 5ml of sucrose solution with the volume percentage of 30% is injected into the concentrated culture medium solution, and after layering occurs, the concentrated culture solution is continuously added, so that the liquid level is up to the position of a pipe orifice of 3 mm; centrifuging, removing the upper culture medium, and gently sucking sucrose solution containing exosomes below by using a syringe; and (3) after collecting the sucrose solution, diluting with PBS buffer solution, placing into an ultrafiltration tube with 100KD, balancing, repeatedly washing with PBS, centrifuging until removing sucrose heavy water solution, and finally collecting concentrated solution which is the exosomes derived from odontogenic stem cells, filtering, sterilizing and storing in a refrigerator at-80 ℃.
2. Identification of exosomes
(1) Transmission Electron Microscope (TEM)
The sample was centrifuged, 10. Mu.l of the sample was pipetted onto a copper mesh, and after standing for several minutes, the excess float of the sample was pipetted using filter paper. After 10 μl of phosphotungstic acid is dripped on a copper mesh and dyed for 1min at room temperature, the floating liquid is sucked by filter paper, after drying for a few minutes, the sample is put under a transmission electron microscope for 80kv-120kv imaging, the physical characteristics of vesicles in the sample, including morphology, diameter size and membrane structure, are identified, the identification result is shown in figure 2, and the exosomes have different morphology sizes under the electron microscope and are similar to circles or ellipsoids.
(2) Exosome particle size analysis
And sucking 10 μl of the extracted exosome sample, diluting the sample by using 1 XPBS buffer solution, performing particle size analysis on the exosome, and determining the particle size of the exosome in the sample to further identify the distribution of the exosome. The experimental process temperature was maintained at 23 ℃ to 37 ℃. As a result, as shown in FIG. 3, it was found from the NTA particle size analysis of the exosomes that the exosomes have a particle size distribution concentrated at 50-150nm.
(3) Western Blot Western Blot detection is carried out on the specific protein markers;
the obtained results are shown in figure 4, and the exosome protein markers TSG101, CD81 and CD63 have clear bands and are positive, so that the method can be proved to be capable of effectively obtaining the odontogenic stem cell exosome.
Example 2:
the application of different inducers in periodontal bone reconstruction is adopted in the embodiment, and the specific steps are as follows:
inducer: radix Puerariae extract, herba Epimedii extract, herba Menthae extract and folium Nelumbinis extract.
The preparation method of the inducer extracts comprises the following steps: 500g of dry kudzuvine root, dry epimedium herb, fresh peppermint and fresh lotus leaf medicinal materials are respectively weighed, and are refluxed for 2 times with ethanol solution with the volume percentage of 70 percent for 2 hours each time, then the extract is concentrated to 20g, and the corresponding inducer is obtained after storage at the temperature of 4 ℃.
Then, mesenchymal stem cell gel, a odontogenic stem cell exosome, bone meal and an inducer are adopted to be applied to periodontal bone reconstruction operation of periodontitis, and the specific steps are as follows: surgical site: is carried out in the south peace, close to the mind, and create and beautify the oral clinic department; case sources 2018-2021; the specific surgical groupings are as follows:
CK group: and selecting periodontal bone retraction cases for operation, applying anti-inflammatory drugs after tooth washing and cleaning, and incising the gingiva after the gingiva is red, swollen and disappeared. Putting bone meal, mesenchymal stem cell culture solution and odontogenic stem cell exosome into the suture (30 cases are produced in total); the mass percent of the mesenchymal stem cell gel of the CK group is 15%, the mass percent of the odontogenic stem cell exosome is 5%, and the mass percent of the bone powder is 80%.
Radix Puerariae inducer group: and selecting periodontal bone retraction cases for operation, applying anti-inflammatory drugs after tooth washing and cleaning, and incising the gingiva after the gingiva is red, swollen and disappeared. Putting bone meal, mesenchymal stem cell culture solution, odontogenic stem cell exosomes and radix puerariae extract into the mixture for stitching (30 cases are taken as a total);
epimedium inducer group: and selecting periodontal bone retraction cases for operation, applying anti-inflammatory drugs after tooth washing and cleaning, and incising the gingiva after the gingiva is red, swollen and disappeared. Putting bone meal, mesenchymal stem cell culture solution, odontogenic stem cell exosomes and epimedium extract into the mixture for stitching (30 cases are taken together);
mint inducer group: and selecting periodontal bone retraction cases for operation, applying anti-inflammatory drugs after tooth washing and cleaning, and incising the gingiva after the gingiva is red, swollen and disappeared. Putting bone meal, mesenchymal stem cell culture solution, odontogenic stem cell exosomes and mint extract into the mixture for stitching (30 cases are taken as a total);
lotus leaf inducer group: and selecting periodontal bone retraction cases for operation, applying anti-inflammatory drugs after tooth washing and cleaning, and incising the gingiva after the gingiva is red, swollen and disappeared. Putting bone powder, mesenchymal stem cell culture solution, odontogenic stem cell exosome and lotus leaf extract, and stitching (30 cases are taken together).
In this example, the mass percentage of mesenchymal stem cell gel of each inducer was 15%, the mass percentage of odontogenic stem cell exosomes was 4%, the mass percentage of bone meal was 80% and the mass percentage of inducer was 1%.
The patient's x-ray film was photographed 6 months after surgery, the heightened bones were measured, the results were both represented by positive and negative standard deviations of the mean, statistical analysis was performed with spss21.0, and significance analysis was performed as needed. The results obtained are shown in Table 1:
TABLE 1 Effect of different inducers on tooth Zhou Gutou increase
| Test item | CK group | Radix Puerariae inducer | Epimedium inducer | Mint inducer | Nelumbo nucifera leaf inducer |
| Heightening (mm) | 2.33±0.11 a | 3.01±0.31 b | 2.35±0.20 a | 3.46±0.21 c | 2.26±0.18 a |
Note that: english a, b, c indicate statistically significant differences (P < 0.05) in the same row comparison.
As can be seen from table 1, the heights of the periodontal bone increases of the epimedium inducer and the lotus leaf inducer are not greatly different from those of the CK group, the differences are not obvious, while Gao Duyuan of the periodontal bone increases of the kudzu root inducer and the peppermint inducer are higher than those of the CK group, and obvious differences are achieved, so that the growth is promoted more effectively, and therefore, not all traditional Chinese medicine inducers can promote the growth of the periodontal bone; the height of periodontal bone increase of the peppermint inducer is higher than that of the kudzu root inducer group, and a significant difference is achieved.
In view of the above results, the applicant has further studied using radix Puerariae and folium Nelumbinis to formulate an inducer, specifically as follows:
surgical site: is carried out in the south peace, close to the mind, and create and beautify the oral clinic department; case sources 2021-2022; the specific operation grouping method is as follows: the periodontal bone recession cases were selected for surgery, after tooth washing and cleaning, anti-inflammatory drugs were applied, the gums were incised and the drugs as shown in Table 2 were placed and then sutured (30 cases were applied to each group) until the gum red swelling disappeared.
Table 2 pharmaceutical combinations of inducers in different ratios
The patient's x-ray film was photographed 6 months after surgery, the heightened bones were measured, the results were both represented by positive and negative standard deviations of the mean, statistical analysis was performed with spss21.0, and significance analysis was performed as needed. The results obtained are shown in Table 3:
TABLE 3 Effect of different drugs on tooth Zhou Gutou increase
| Test item | Group 1 | Group 2 | Group 3 | Group 4 | Group 5 |
| Heightening (mm) | 3.13±0.01 a | 3.10±0.22 a | 4.18±0.17 b | 3.08±0.15 a | 3.05±0.18 a |
Note that: english a, b indicates that the same row comparison has statistically significant differences (P < 0.05).
As can be seen from table 3, the effect of the root of kudzu vine inducer and the mint inducer on periodontal bone elevation was best significantly higher (best) than that of the single-thin-charge inducer of table 2 when the mass ratio of the root of kudzu vine inducer to the mint inducer was 1:3, while the other mass ratios were slightly higher than Shan Gegen inducer than that of the single inducer, indicating that the root of kudzu vine inducer and the mint inducer had significant effects only when the mass ratio was 1:3 when used in combination, and the effect of the other mass ratios on bone elevation was not great.
The above study results show that: although it is reported in the prior art that the traditional Chinese medicine preparation can promote osteogenic differentiation as an inducer, in the practical application process, bone growth is not necessarily promoted, and it is possible that the components in the traditional Chinese medicine preparation have the effect of promoting osteogenic differentiation and inhibiting cell growth, and the dosage is required to be strictly controlled; it is also possible that the present application may be applied to the indications of periodontitis, and that certain traditional Chinese medicine agents, although promoting osteogenic differentiation, do not have an effective anti-inflammatory and cell differentiation promoting effect on specific conditions.
The bone meal of the application is purchased from south Beijing Yiterbo medical science and technology development Co.
The x-ray images of some cases of the application are shown in figures 5-12:
case 1:
zhang Jie 64, periodontitis is diagnosed at hospital arrival; at the time of day 12 and 8 of 2021, the teeth were photographed as shown in fig. 4, and after surgery and treatment with the bone reconstruction drug of example 2 group 3, the teeth were photographed as shown in fig. 5 after 6 months; the dental film showed a 4.0mm increase in periodontal bone.
Case 2:
zheng Weixiong 73 years old, periodontitis was diagnosed at hospital arrival; at the time of day 1 and 2 of 2022, the patient had a dental film as shown in fig. 6, and after surgery and treatment with the bone reconstruction drug of example 2 group 3, the patient had a dental film as shown in fig. 7 after 6 months; the dental film showed 3.9mm increase in periodontal bone.
Case 3:
the 45-year old Zhiti is diagnosed as periodontitis when the hospital comes; at the time of 20 days of 2021, 10 months, the doctor took the teeth as shown in fig. 8, and 6 months after surgery and the addition of the bone reconstruction drug of example 2 group 3, the doctor took the teeth as shown in fig. 9; the dental film showed a 4.1mm increase in periodontal bone.
Case 4:
li Zhiyang 47 years old, periodontitis was diagnosed at hospital arrival; at the time of day 4 and 10 of 2021, the teeth were photographed as shown in the left panel of fig. 10, and after surgery and the addition of the bone reconstruction drug of example 2 group 3, the teeth were photographed as shown in the right panel of fig. 10 after 6 months; the dental film showed a 4.2mm increase in periodontal bone.
Case 5:
yu Guo 40, periodontitis is diagnosed at hospital arrival; at the time of day 3 of 6 of 2021, the teeth were photographed as shown in the left panel of fig. 11, and after surgery and the addition of the bone reconstruction drug of example 2 group 3, the teeth were photographed as shown in the right panel of fig. 11 after 6 months; the dental film showed a 4.2mm increase in periodontal bone.
Example 3:
the influence of exosomes on periodontal bone remodeling was studied in this example, as follows:
periodontal bone reconstruction procedures are performed by the method disclosed in reference 202110311551.X, as follows
CK group: and selecting periodontal bone retraction cases for operation, applying anti-inflammatory drugs after tooth washing and cleaning, and incising the gingiva after the gingiva is red, swollen and disappeared. Placing mesenchymal stem cell gel 19%, bone powder 80% and inducer 1% for suturing (20 cases are taken together); wherein the inducer is radix Puerariae extract (the preparation method of the extract is shown in example 2);
exosome group: and selecting periodontal bone retraction cases for operation, applying anti-inflammatory drugs after tooth washing and cleaning, and incising the gingiva after the gingiva is red, swollen and disappeared. Placing 15% of mesenchymal stem cell gel, 4% of odontogenic stem cell exosome, 80% of bone meal and 1% of inducer for suturing (20 cases are produced); wherein the inducer is radix Puerariae extract (the preparation method of the extract is shown in example 2)
The patient's x-ray film was photographed 6 months after surgery, the heightened bones were measured, the results were both represented by positive and negative standard deviations of the mean, statistical analysis was performed with spss21.0, and significance analysis was performed as needed. The results obtained are shown in Table 4:
TABLE 4 influence of exosomes on tooth Zhou Gutou increase
| Test item | CK group | Exosome group |
| Heightening (mm) | 2.54±0.11 a | 3.10±0.02 b |
Note that: english a, b indicates that the same row comparison has statistically significant differences (P < 0.05).
From the above table, the bone elevation effect of periodontal bone is better than that of the control group without exosomes after exosomes are added, which indicates that the odontogenic exosomes have the effect of promoting bone differentiation and can promote the bone formation of periodontal bone; the bone-increasing effect of the CK group was slightly lower than that of patent 202110311551.X, indicating that bone-meal content was also a factor affecting periodontal bone formation.
From the comprehensive examples 1-3, the optimum formulation of the drug for promoting periodontal bone remodeling is as follows: 15% of mesenchymal stem cell gel, 4% of odontogenic stem cell exosomes, 80% of bone meal and 1% of inducer; wherein the inducer is prepared by mixing radix Puerariae extract and herba Menthae extract at a mass ratio of 1:3.
(1) Inoculating dental pulp stem cells into a culture dish or a culture bottle, discarding old culture medium when the growth of the cells is fused to 80%, washing 3 times by PBS, and adding a basal culture medium;
(2) After culturing for 24-48 hours, collecting the cell culture solution;
(3) DMEM washed with PBS to remove dead cells and replaced with 10% fbs;
(4) Continuously collecting about 500ml of cell culture solution, and sub-packaging into 50ml centrifuge tubes;
centrifuging to remove macromolecular vesicles, concentrating in a 100KD ultrafiltration tube, discarding liquid with a height of 1.5cm from the bottom of the centrifugation tube, and centrifuging at 5000g at 4deg.C for 10min to obtain culture medium concentrate; 5ml of sucrose solution with the volume percentage of 30% is injected into the concentrated culture medium solution, and after layering occurs, the concentrated culture solution is continuously added, so that the liquid level is up to the position of a pipe orifice of 3 mm; centrifuging, removing the upper culture medium, and gently sucking sucrose solution containing exosomes below by using a syringe; and (3) after collecting sucrose solution, diluting with PBS buffer solution, placing into an ultrafiltration tube with 100KD, balancing, repeatedly washing with PBS, centrifuging until removing sucrose heavy water solution, and finally collecting concentrated solution which is an exosome of dental stem cells, filtering, sterilizing, and storing in a refrigerator at-80 ℃.
In conclusion, compared with the prior art, the medicine provided by the application can effectively reconstruct periodontal bones, and deep research on inducers shows that: the kudzu root extract and the mint extract can be used as inducers to improve the reconstruction effect on periodontal bones.
The above examples only represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the present application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of the application should be assessed as that of the appended claims.
Claims (5)
1. A medicament for promoting periodontal bone reconstruction by utilizing stem cell exosomes, which is characterized by comprising the following components in percentage by mass: 15% of mesenchymal stem cell gel, 4% of odontogenic stem cell exosomes, 80% of bone meal and 1% of inducer; the inducer is radix Puerariae extract and herba Menthae extract;
the radix puerariae extract and the mint extract are ethanol extracts;
the mass ratio of the kudzuvine root extract to the mint extract is 1:3.
2. The drug for promoting periodontal bone remodeling by stem cell exosomes according to claim 1, wherein the ethanol of the ethanol extract is 70% ethanol solution by volume.
3. The medicine for promoting periodontal bone reconstruction by utilizing stem cell exosomes according to claim 1, wherein the specific extraction method of the kudzuvine root extract and the peppermint extract is as follows: 500g of dried kudzuvine root and fresh peppermint are respectively weighed, and are refluxed for 2 times by using 70 percent ethanol solution for 2 hours each time, then the extract is concentrated to 20g, and the corresponding extract is obtained after being stored at the temperature of 4 ℃.
4. A medicament for promoting periodontal bone remodeling using stem cell exosomes according to claim 1, wherein the odontogenic stem cell exosomes markers are TSG101, CD81 and CD63 proteins.
5. A medicament for promoting periodontal bone remodeling using stem cell exosomes according to claim 1, wherein the preparation method of the odontogenic stem cell exosomes is as follows:
(1) Inoculating dental pulp stem cells into a culture dish or a culture bottle, discarding old culture medium when the growth of the cells is fused to 80%, washing 3 times by PBS, and adding a basal culture medium;
(2) After culturing for 24-48 hours, collecting the cell culture solution;
(3) DMEM washed with PBS to remove dead cells and replaced with 10% fbs;
(4) Continuously collecting about 500ml of cell culture solution, and sub-packaging into 50ml centrifuge tubes; (5) Centrifuging to remove macromolecular vesicles, concentrating in a 100KD ultrafiltration tube, discarding liquid with a height of 1.5cm from the bottom of the centrifugation tube, and centrifuging at 5000g at 4deg.C for 10min to obtain culture medium concentrate; 5ml of sucrose solution with the volume percentage of 30% is injected into the concentrated culture medium solution, and after layering occurs, the concentrated culture solution is continuously added, so that the liquid level is up to the position of a pipe orifice of 3 mm; centrifuging, removing the upper culture medium, and gently sucking sucrose solution containing exosomes below by using a syringe; and (3) after collecting sucrose solution, diluting with PBS buffer solution, placing into an ultrafiltration tube with 100KD, balancing, repeatedly washing with PBS, centrifuging until removing sucrose heavy water solution, and finally collecting concentrated solution which is an exosome of dental stem cells, filtering, sterilizing, and storing in a refrigerator at-80 ℃.
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| JP2010260794A (en) * | 2009-04-30 | 2010-11-18 | Lion Corp | Alveolar bone absorption inhibitor |
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| CN112843339A (en) * | 2021-03-24 | 2021-05-28 | 吴涛 | Application of mesenchymal stem cells in preparation of periodontal bone reconstruction medicine |
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| CN102625866A (en) * | 2009-04-01 | 2012-08-01 | 高露洁-棕榄公司 | Menthol-derivative compounds and use thereof as oral and systemic active agents |
| JP2010260794A (en) * | 2009-04-30 | 2010-11-18 | Lion Corp | Alveolar bone absorption inhibitor |
| CN112843339A (en) * | 2021-03-24 | 2021-05-28 | 吴涛 | Application of mesenchymal stem cells in preparation of periodontal bone reconstruction medicine |
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