CN112843339A - Application of mesenchymal stem cells in preparation of periodontal bone reconstruction medicine - Google Patents
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- 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
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Abstract
The invention discloses an application of mesenchymal stem cells in preparation of a periodontal bone reconstruction drug, wherein the drug comprises the following components in percentage by mass: 14.5 percent of mesenchymal stem cell gel, 85 percent of allogeneic bone powder and 0.5 percent of inducer. The invention applies the mesenchymal stem cells to the preparation of the periodontal bone reconstruction medicine, and can promote the repair and reconstruction of the periodontal bone.
Description
Technical Field
The invention relates to the technical field of alveolar bone repair, in particular to application of mesenchymal stem cells in preparation of an alveolar bone defect repair medicine.
Background
Periodontitis is a chronic inflammation that invades the gingiva and periodontal tissue, and is a destructive disease mainly characterized by a group characterized by the formation of periodontal pockets and inflammation of pocket walls, periodontal bone loss, and tooth loosening, which are malignant diseases of the oral cavity.
At present, the treatment method of periodontitis is firstly a local treatment, mainly removing supragingival calculus and subgingival calculus, and then removing diseased cementum containing a large amount of bacterial toxins from periodontal pockets by scraping, and after these treatments, gingival redness and swelling can be resolved, and gingival bleeding and periodontal pocket purulence can be disappeared. The systemic treatment of periodontitis is currently effective in removing dental calculus and medicines locally. The tinidazole medicine is used for the whole body, and no good method is provided for reconstructing the missing periodontal bone at present.
Therefore, how to apply the mesenchymal stem cells to the preparation of periodontal bone reconstruction drugs is a problem which needs to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of the above, the mesenchymal stem cells are applied to the preparation of the periodontal bone reconstruction drug, so that the repair and reconstruction of the periodontal bone can be promoted.
In order to achieve the purpose, the invention adopts the following technical scheme:
application of mesenchymal stem cells in preparing periodontal bone reconstruction medicines.
The medicine for reconstructing periodontal bone comprises the following components in percentage by mass: 14.5 percent of mesenchymal stem cell gel, 85 percent of allogeneic bone powder and 0.5 percent of inducer.
According to the technical scheme, after the obtained mesenchymal stem cells are subjected to induction amplification, the mesenchymal stem cells MSCS are induced to generate cells suitable for tissue growth in a culture solution, the cells are mixed with allogeneic bone powder, an inducer is placed into the cells, periodontal flap surgery is performed in an operation area and placed at a bone defect to repair the bone defect, x-ray examination is performed after six months, cementum lines at the x-ray visible bone defect grow again, the bone level is increased, and the trabecula is recovered.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 accompanying drawing is a microscopic observation of mesenchymal stem cells;
FIG. 2 is a diagram showing the expression of CD34 in fluorescence detection with 200-fold ocular lens;
FIG. 3 is a diagram showing the expression of CD45 in fluorescence detection with 200-fold ocular lens;
FIG. 4 is a diagram showing the expression of CD73 in fluorescence detection using a 200-fold eyepiece;
FIG. 5 is a diagram showing the expression of CD90 in fluorescence detection using a 200-fold eyepiece;
FIG. 6 is a diagram showing the expression of CD105 in the 200-fold ocular fluorescence detection;
FIG. 7 is a graph showing the results of flow cytometry measurement of CD73 concentration;
FIG. 8 is a graph showing the results of flow cytometry measurement of CD90 concentration;
FIG. 9 is a graph showing the results of flow cytometry for CD105 concentration;
FIG. 10 is a diagram showing the results of HLA-DR expression detection by flow cytometry;
FIG. 11 is a graph showing the results of flow cytometry detection of CD 34;
FIG. 12 is a graph showing the results of flow cytometry detection of CD 45;
FIG. 13 is a graph showing the results of flow cytometry detection of CD 14;
FIG. 14 is a graph showing the results of flow cytometry detection of CD 19;
FIG. 15 is a graph illustrating preoperative characterization of a condition in a patient;
figure 16 is a graph of the effect of mesenchymal stem cells after treatment.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 stem cell enrichment.
Experimental reagent
Physiological saline, PBS buffer solution, blood cell counting reagent, mesenchymal stem cell culture medium, serum-free additive, polyformaldehyde fixing agent, TritonX-100, BSA, DAPI, HLA-DR antibody, CD14 antibody, CD19 antibody, CD34 antibody, CD45 antibody, CD73 antibody, CD90 antibody, CD105 antibody, fluorescence-labeled goat anti-mouse antibody, anti-crash incubation solution and anti-crash sealed tablet which are all purchased from HYCLONE company;
experiment consumables: blood collection tube, blood collection needle, 96-hole culture plate, 24-hole culture plate, sterile cover glass, glass slide, pipette, centrifuge tube, Pasteur pipette, sterile suction head, EP tube, medical variable speed centrifuge, hemocyte analyzer, CO2Incubator, fluorescence inversion microscope, super clean bench, micropipetteMicro electronic balance, flow cytometer
Experimental methods
Enrichment:
PRF cell layer separation and collection venous peripheral blood 10mL (ordinary negative pressure tube), put into the centrifuge of variable speed within three minutes, set up the preparation procedure. The centrifugation procedure is divided into four phases. After the variable speed centrifugation is finished, in an aseptic operation table, the blood collection tube is opened, the jelly-like PRF is taken out, the blood collection tube is transferred to a disinfection vessel, the upper layer of faint yellow colloid is cut off, the jelly red blood cells are simply washed by 5mL of physiological saline, and the colloid is reserved.
And (4) culture increase and passage:
the cells were subcultured in a mesenchymal stem cell-dedicated medium (purchased from HYCLONE) and harvested at p4 passages.
And (3) carrying out immunofluorescence staining detection:
preparing single cell suspension from cells to be detected, inoculating the cells onto sterile cover glass placed in a 24-well plate in advance, and placing the cover glass on CO2Culturing for 48 hours in an incubator to allow the cells to be detected to adhere to the wall. Discarding the cell culture medium, washing twice with PBS, removing nonadherent cells such as erythrocytes, adding paraformaldehyde solution with final concentration of 4%, fixing at room temperature for 30min, and washing the cover glass with PBS for 2 times, each for 3 min. The permeabilization solution TritonX-100 was added to a 24-well plate, permeabilized for 10min at room temperature, washed 2 times with PBS, 3min each time. PBS containing 5% BSA was added to the 24-well plate, blocked at room temperature for 30min, and the liquid was discarded. Primary antibody was diluted with PBS at a certain ratio and added to each well to be tested at 200. mu.L per well, incubated at room temperature for 2h, the primary antibody was aspirated off, and washed 3 times with PBS for 5min each time. Diluting the secondary antibody coupled with the fluorescent dye according to a proper proportion, incubating for 30min in a dark condition by 200 mu L per well, and absorbing and discarding the secondary antibody. After washing the slide with PBS, 100. mu.L of DAPI with appropriate concentration is added, the cell nucleus is stained for 10min in the dark, the staining solution is discarded, after washing with PBS, the cover glass is taken out, the residual liquid on the cover glass is removed by using filter paper, a drop of anti-quenching mounting agent is dropped in the middle of the slide, and then the cover glass is reversely covered on the slide for observation under a mirror. The mesenchymal stem cells visible under the mirror are shown in figure 1; the expression of CD34 in the 200-fold ocular fluorescence detection is shown in FIG. 2; 200-fold Ocular immunofluorescence CD45 expression As shown in FIG. 3Shown in the specification; CD73 expression was detected by 200-fold ocular immunofluorescence as shown in figure 4; the 200-fold ocular CD90 immunofluorescence detection expression is shown in figure 5; CD105 expression was detected by 200-fold immunofluorescence as shown in figure 6;
flow cytometry detection of cellular markers:
collecting single cell suspension to make its cell density be 0.5-1 × 107mL, and a total volume of greater than 800. mu.L. After gently pipetting and mixing well, the cell suspension is subpackaged into EP tubes in a volume of 100 mu L/tube, fluorescence-labeled HLA-DR, CD14, CD19, CD45, CD73, CD90 and CD105 antibodies are respectively added into each tube, and gently pipetting and mixing well. And covering the sample tube added with the antibody with tin box paper, placing the sample tube in a dark place, and incubating at normal temperature for 20-30 min. After the incubation is completed, the sample tube is centrifuged at 400g for 5min at 4 ℃, the supernatant containing the antibody is carefully aspirated, 500 μ L of PBS is added for resuspension, and the sample tube is placed at 4 ℃ in a dark place for detection. Mixing by gentle blowing before detection, detecting with flow cytometer, and detecting CD73 concentration as shown in FIG. 7; CD90 concentration measurements are shown in fig. 8; CD105 concentration measurements are shown in figure 9; HLA-DR expression results are shown in FIG. 10; CD34 results are shown in fig. 11; CD45 results are shown in fig. 12; CD14 results are shown in fig. 13; CD19 results are shown in fig. 14;
analysis of results
According to the detection standard of the international union of cell therapy, the mesenchymal stem cells express CD73, CD90 and CD 105. Therefore, it was found that the culture medium contains mesenchymal stem cells and many growth-assisting cells by flow cytometry analysis. (all the results are provided by the detection reports of national drug gene and protein screening laboratories).
Clinical cases
The case of bone recession and shrinkage of periodontal is selected for operation, and after tooth washing and cleaning, the medicine for eliminating inflammation is used to cut the gingiva until the redness and swelling of gingiva disappear. Placing bone powder, mesenchymal stem cell culture solution and inducer, and suturing.
3x ray film observation. After 6 months, the patient was photographed with a control x-ray film and the increased bone was measured.
FIG. 15 shows the view of periodontal bone defects of upper and lower teeth before operation; the X-ray results at 6 months after surgery are shown in figure 16.
Results are expressed as mean positive and negative standard deviations, statistical analysis is performed with a sps 21.0, t-test analysis is performed as required, p is less than 0.05, and statistical results show that 35 customers have an average periodontal increase of three millimeters.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. Application of mesenchymal stem cells in preparing periodontal bone reconstruction medicines.
2. The medicine for reconstructing periodontal bone comprises the following components in percentage by mass: 14.5 percent of mesenchymal stem cell gel, 85 percent of allogeneic bone powder and 0.5 percent of inducer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116350676A (en) * | 2023-04-06 | 2023-06-30 | 吴涛 | Medicine for promoting periodontal bone reconstruction by stem cell exosome |
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| 吴中明: ""TGF-β3与牙髓干细胞联合Bio-oss骨粉在动物骨缺损修复中的作用研究"", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116350676A (en) * | 2023-04-06 | 2023-06-30 | 吴涛 | Medicine for promoting periodontal bone reconstruction by stem cell exosome |
| CN116350676B (en) * | 2023-04-06 | 2023-11-28 | 吴涛 | Medicine for promoting periodontal bone reconstruction by stem cell exosome |
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