CN112972496A

CN112972496A - Application of mesenchymal stem cells in preparation of alveolar bone defect repair medicine

Info

Publication number: CN112972496A
Application number: CN202110310675.6A
Authority: CN
Inventors: 吴涛
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-03-24
Filing date: 2021-03-24
Publication date: 2021-06-18

Abstract

The invention discloses application of mesenchymal stem cells in preparation of a medicine for repairing alveolar bone defects. Compared with the prior art, the invention uses simple stem cells as bone graft materials, does not use GBR membrane covering technology, has no other bone filling materials compared with the traditional GBR operation, can form bones without any membrane covering, and subverts the GBR concept.

Description

Application of mesenchymal stem cells in preparation of alveolar bone defect repair medicine

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

Alveolar bone, commonly known as alveolar process, refers to the part of upper and lower jaws surrounding and supporting tooth roots, and is clinically called alveolar bone defect, which is caused by poor oral hygiene environment, malocclusion, lumps or congenital factors, external factors such as traffic accidents, wounds and the like, and the integrity of the alveolar bone is damaged. Under normal conditions, the alveolar bone wraps around the tooth root, and the defect can cause tooth loosening and falling off, such as alveolar bone loss caused by trauma, malocclusion and facial appearance change.

At present, GRB technology is generally applied to medical alveolar bone repair, common GBR operation requirements are to cover with a film and fill materials with bones, a bone growth space is kept airtight, mucosal epithelial cells are ensured not to grow into bone tissues, bone defects can be repaired at the same time, mesenchymal stem cells are more and more generally applied in the field of oral regenerative medicine, but clinical research supporting the effect of the mesenchymal stem cells in GBR is still very limited. The mesenchymal stem cells have more data for soft tissue regeneration, but no research has proved that the mesenchymal stem cells have bone regeneration potential for alveolar bone defects.

Therefore, how to apply the mesenchymal stem cells to the preparation of the alveolar bone defect repair medicine 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 alveolar bone defect repair medicine, and are independently applied to the alveolar bone repair for the first time, so that the regeneration of the alveolar bone is promoted.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of the mesenchymal stem cells in preparing the alveolar bone defect repairing medicine.

A medicine for repairing alveolar bone defects comprises a mesenchymal stem cell gel and an inducer, wherein the mass ratio of the mesenchymal stem cell gel to the inducer is 99: 1.

According to the technical scheme, compared with the prior art, the bone grafting device uses simple stem cells as a bone grafting material, does not use a GBR membrane covering technology, does not have other bone filling materials compared with the traditional GBR operation, can form bones without any membrane covering, and adopts the principle of GBR to isolate the cells by using the membrane and grow bone powder in a bone cavity. The invention uses the cell regeneration principle, differentiates stem cells into osteoblasts, fills bone cavities and overturns the GBR concept.

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 fluorescence detection with 200-fold ocular lens;

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;

FIG. 16 is a graph of patient characterization;

FIG. 17 is a view of a capsular bag being removed during surgery;

FIG. 18 is a drawing of a preoperative x-ray representation of 21 root resorption and bone destruction;

FIG. 19 is a graph showing CT results of a defect cheek bone after nine months of operation;

FIG. 20 is a graph showing CT results of a defect buccal bone at nine months after the operation;

FIG. 21 is a graph showing CT results of a defect buccal bone at nine months after the operation;

FIG. 22 is a graph showing CT results of a defect buccal bone at nine months after the operation;

FIG. 23 is a graph showing CT results of a defect buccal bone at nine months after the operation;

FIG. 24 is a graph showing CT results of a nine-month defect buccal bone after surgery;

FIG. 25 is a graph showing CT results of a defect buccal bone at nine months after the operation;

FIG. 26 is a graph showing CT results of a defect buccal bone at nine months after the operation.

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 tubes, blood collection needles, 96-well culture plates, 24-well culture plates, sterile cover slips, glass slides, pipettes, centrifuge tubes, pasteur pipettes, sterile suction heads, EP tubes, medical variable speed centrifuges, hemocyte analyzers, CO2 incubator, fluorescence inverted microscopes, superclean platforms, micropipettes, micro electronic balances, flow cytometers;

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 to a disinfection vessel, the upper layer of faint yellow colloid is cut and removed, 5mL of physiological saline is used for simply cleaning the red blood cells of the jelly, 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; the 200-fold ocular immunofluorescence CD45 expression is shown in figure 3; 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 finished, the sample tube is centrifuged for 5min at 400g at 4 ℃,the supernatant containing the antibody was carefully aspirated, resuspended in 500. mu.L of PBS, and placed at 4 ℃ in the dark for assay. 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

Introduction of medical records: wang xx, male, 17 years old, will be diagnosed half a year because of the bone pressure pain of maxillary sinus. And (4) checking: 21 coronal folds, medullary perforation, percussion pain (++), loose (+), 21, 22, 23, 24, 25 pulpal heat test negative. ct showed 21, 22, 23, 24, 25 giant apical cysts, 3.5x3mm soft, tenderness. Ct indicates maxillary solid floor destruction.

And (3) diagnosis: 1, 21, 22, 23, 24, 25 dead pulp tooth 2.21, 22, 23, 24, 25 area giant apical cyst 321 crown

The operation process comprises the following steps: cutting the lower edge of the local anesthesia along the crest of the dental alveolus, stripping off mucosa, removing cyst, scraping off periosteum, washing with physiological saline and tinidazole antibiotic solution, and completing filling treatment of dental cement and filling paste with clear and super filling of root canal in situ. The maxillary sinus floor was found to be damaged, 8X 10mm in area. Perforating mucosa of maxillary sinus. Lax maxillary sinus inner membrance, with absorbable suture around lax maxillary sinus mucosa meshwork, because the sinus mucosa is extremely thin, can not pull by force, still leave the clearance about 1mm because the operation cavity is very big, cavity upper portion hone lamella destroys, has the gap, consequently can not use traditional GBR technique, fills out bone meal, the tectorial membrane. The cavities can only be filled with mesenchymal stem cell gel (obtained by subjecting plasma to variable speed centrifugation, wherein fibrin in the plasma forms gel after red blood cells are filtered out) and inducer (SDF1, also known as stromal cell derived factor, available from HYCLONE) at a mass ratio of 99: 1. The external mucosa is sutured.

Before surgery, the patient is shown in fig. 15; cysts have rotted through the bone as shown in fig. 16; the enucleated giant jaw cyst is shown in fig. 17; preoperative x-ray visualization 21 root resorption and bone destruction, as shown in fig. 18;

and (3) operation results: after 9 months, new bone was formed throughout the cavity. New bone wraps and cementum lines were visible around the original bare root tip. CT showed a 3mm height of new bone of corroded bone and the appearance of bone regeneration, see fig. 19-26;

the Guangxi institute for medical science and information research report demonstrated that the invention of Wutao was not done in literature reports around the world.

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

1. The application of mesenchymal stem cells in the preparation of alveolar bone defect repair drugs.

2. A medicine for repairing alveolar bone defect, characterized in that it comprises a mesenchymal stem cell gel and an inducer, and the mass ratio of the mesenchymal stem cell gel and the inducer is 99:1.