PDGFRA + odontogenic mesenchymal stem cell polymer as well as preparation method and application thereof

Abstract

The invention is suitable for the technical field of biomedicine, and provides a PDGFRA + odontogenic mesenchymal stem cell polymer and a preparation method and application thereof.The preparation method comprises the steps that odontogenic mesenchymal stem cells obtained through separation are labeled with magnetic bead-antibodies, the PDGFRA + odontogenic mesenchymal stem cells are obtained through separation, induction culture is conducted through a polymer induction culture medium, and the PDGFRA + odontogenic mesenchymal stem cell polymer is obtained. Obtaining a PDGFRA + odontogenic mesenchymal stem cell polymer; the polymer induction culture medium is prepared from an alpha-MEM culture medium, ascorbic acid, L-glutamine, penicillin and streptomycin. The method is simple in step, convenient to operate, low in cost, short in preparation period and easy to industrialize, the obtained polymer has a good periodontal bone tissue regeneration-angiogenesis promoting coupling function, endothelial cells are promoted to form lumens through exchange with host endothelial cells, a feasible strategy is provided for regeneration treatment of bone tissue injuries and degenerative diseases, and the polymer has a good application prospect. The clinical application prospect is better.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology and in particular to a PDGFRA + Dental mesenchymal stem cell aggregates and preparation method and application thereof. Background Art

[0002] Periodontal bone tissue defect is a common periodontal disease in clinical practice. Severe periodontal bone tissue defect may even lead to tooth loss, which significantly impairs the function of the masticatory system, affects facial aesthetics, and may even cause digestive system problems and psychological disorders. Therefore, it is crucial to explore effective periodontal bone regeneration solutions. However, current regenerative therapies, including barrier membranes, transplant materials, growth factors, or their combination, are still unstable in repairing missing periodontal tissues, and how to promote the repair and regeneration of periodontal tissues still faces major challenges.

[0003] Periodontal bone regeneration is a complex biological process involving the interaction of multiple cell types and signaling pathways. + Cells have previously been identified as mesoderm, somites, and mesenchyme progenitors. In tooth formation, studies have revealed that PDGFA and PDGFRA act as autocrine mechanisms to mediate continuous epithelial-mesenchymal interactions during tooth development, which is essential for tooth size and cusp morphogenesis. Recently, further studies have found that PDGFRA + and PDGFRB + Cells differentiate into different cell lineages in adult mouse incisors by accepting PDGF ligands deposited in the MSC region from the arteries. However, specific types of PDGFRA + The application of dental stem cell aggregates in promoting periodontal bone tissue regeneration is still unclear. + The cellular properties and how the tooth development microenvironment regulates these mesenchymal progenitor cells need to be further elucidated.

[0004] Periodontal tissue development originates from the condensed dental follicle tissue during tooth development. Inspired by this natural process, constructing specific dental follicle stem cell aggregates has become a key aspect to improve the efficiency of periodontal tissue regeneration. + It is still unclear how dental mesenchymal stem cell aggregates are constructed and how they promote periodontal tissue regeneration. Summary of the invention

[0005] The purpose of the present application embodiment is to provide a PDGFRA + Method for preparing dental mesenchymal stem cell aggregates, aiming to obtain PDGFRA with good functions of promoting periodontal bone tissue regeneration and H-type vascular coupling+ Dental mesenchymal stem cell aggregates.

[0006] The embodiment of the present application is implemented as follows: a PDGFRA + A method for preparing a dental mesenchymal stem cell aggregate comprises:

[0007] The isolated dental mesenchymal stem cells were labeled with magnetic beads and antibodies, and PDGFRA was sorted. + Dental mesenchymal stem cells;

[0008] The PDGFRA + Dental mesenchymal stem cells were induced and cultured to obtain PDGFRA + Dental mesenchymal stem cell aggregates;

[0009] Wherein, the polymer induction medium is composed of the following components: α-MEM medium, ascorbic acid, L-glutamine, penicillin and streptomycin.

[0010] The present application embodiment also provides a PDGFRA + Dental mesenchymal stem cell aggregates, the PDGFRA + Dental mesenchymal stem cell aggregates are composed of the above-mentioned PDGFRA + The dental mesenchymal stem cell aggregates are prepared by the preparation method.

[0011] The present application also provides a PDGFRA + Application of dental mesenchymal stem cell aggregates in the preparation of drugs for the treatment of periodontal tissue defects.

[0012] The present application also provides a drug for treating periodontal tissue defects, the drug comprising the above-mentioned PDGFRA + Dental mesenchymal stem cell aggregates, and pharmaceutically acceptable excipients and carriers.

[0013] The present application embodiment provides PDGFRA + The preparation method of dental mesenchymal stem cell aggregates has simple steps, convenient operation, low equipment use cost, short preparation cycle, and easy industrialization; the PDGFRA prepared in this application + Dental mesenchymal stem cell aggregates have a good function of promoting periodontal bone tissue regeneration-angiogenesis coupling. Their specific role is to promote endothelial cell lumen formation through communication with host endothelial cells, providing a feasible strategy for the regenerative treatment of bone tissue damage and degenerative lesions, and have more clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A flowchart of the experimental design for implanting dental mesenchymal stem cells (DFSCs) aggregates into periodontal bone defects for periodontal bone tissue regeneration provided in the embodiments of the present application;

[0015] Figure 2 A diagram showing the Micro-CT analysis results of periodontal bone tissue regeneration in rats provided in the examples of the present application;

[0016] Figure 3 H&E staining of the periodontal bone defect area provided in the examples of the present application;

[0017] Figure 4 This is an immunofluorescence staining image of the periodontal bone defect area provided in the examples of the present application. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0019] The present application embodiment provides a PDGFRA + A method for preparing a dental mesenchymal stem cell aggregate comprises:

[0020] The isolated dental mesenchymal stem cells were labeled with magnetic beads and antibodies, and PDGFRA was sorted. + Dental mesenchymal stem cells;

[0021] The PDGFRA + Dental mesenchymal stem cells were induced and cultured to obtain PDGFRA + Dental mesenchymal stem cell aggregates;

[0022] Wherein, the polymer induction medium is composed of the following components: α-MEM medium, ascorbic acid, L-glutamine, penicillin and streptomycin.

[0023] In the embodiments of the present application, dental mesenchymal stem cells include apical dental papilla stem cells (SCAP) and dental follicle stem cells (DFSCs). Although the following specific embodiments of the present application only take dental follicle stem cells as an example, this should not limit the scope of protection of the present application. Specifically, the effects of preparing stem cell aggregates using the above-mentioned dental mesenchymal stem cells as raw materials and according to the method provided in the present application are equivalent, and the effects of the generated stem cell aggregates in promoting periodontal bone tissue regeneration-angiogenesis and other aspects are also equivalent. Those skilled in the art can select the type of dental mesenchymal stem cells according to actual needs during specific implementation.

[0024] In the examples of the present application, the polymer induction medium is prepared according to the following steps:

[0025] Ascorbic acid, L-glutamine, penicillin and streptomycin were added to α-MEM medium. The concentration of ascorbic acid was 0-100 μg / mL, the concentration of L-glutamine was 0.5-10 mM, the concentration of penicillin was 100 U / mL, and the concentration of streptomycin was 100 g / mL.

[0026] Optionally, the polymer induction medium is prepared according to the following steps:

[0027] Ascorbic acid, L-glutamine, penicillin and streptomycin were added to α-MEM medium. The concentrations of ascorbic acid, L-glutamine, penicillin, and streptomycin were 50 μg / mL, 2 mM, 100 U / mL, and 100 U / mL, respectively.

[0028] In the present application example, the dental mesenchymal stem cells isolated were labeled with magnetic beads-antibodies and PDGFRA was sorted. + The steps of dental mesenchymal stem cells include:

[0029] The dental mesenchymal stem cells separated and subcultured were then treated with enzyme digestion to obtain a single cell suspension;

[0030] The single cell suspension is incubated with a biotin-labeled CD140a monoclonal antibody, the resulting cells are combined with avidin positive selection beads, washed, and placed in a magnetic field to sort out PDGFRA + Dental mesenchymal stem cells.

[0031] Optionally, the single cell suspension was incubated with 2uL of biotin-labeled CD140a monoclonal antibody at room temperature for 10 min, the resulting cells were bound to avidin positive selection beads, washed with 3% FBS-PBS separation buffer, and placed in a magnetic field to sort out PDGFRA + Dental mesenchymal stem cells.

[0032] In the examples of the present application, the induction culture conditions are: culture at 37° C. and 5% CO 2 for 7-14 days.

[0033] The following specific examples are used to describe PDGFRA. + Dental mesenchymal stem cell aggregates and their preparation methods and applications are described in detail as follows. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are all commercially available unless otherwise specified.

[0034] The test materials used in the following examples are all common commercial products and can be purchased on the market. The purchase channels of some major reagents and materials are as follows: α-MEM culture medium (Gibco: C12571500bt); ascorbic acid (02100769-CF, MP Biomedical, USA); penicillin and streptomycin (15070063, both from Invitrogen, USA); L-glutamine (35050061, Invitrogen, USA).

[0035] Example: PDGFRA + Preparation process of dental mesenchymal stem cell aggregates and their application in periodontal regeneration

[0036] like Figure 1 As shown, the specific steps include:

[0037] (1) Magnetic bead separation of PDGFRA + Dental mesenchymal stem cells

[0038] a. Isolate and obtain dental follicle tissue, and culture dental follicle stem cells according to existing methods;

[0039] b. Dental follicle stem cells (P1) after passage are used to isolate PDGFRA + Dental mesenchymal stem cells;

[0040] c. After trypsin digestion, a single cell suspension was prepared and incubated with 2uL of biotin-labeled CD140a (i.e., PDGFRA) monoclonal antibody (13-1401-80, Thermofisher, USA) at room temperature for 10 minutes;

[0041] d. Sort cells with MagniSort according to the manufacturer's instructions. TM Streptavidin positive selection beads (MSPB-6003-74, Thermofisher, USA) binding;

[0042] e. After washing with 3% FBS-PBS separation buffer, the cells were placed in a magnetic field to separate positive and negative cell populations (PDGFRA + Dental mesenchymal stem cells, PDGFRA - Dental mesenchymal stem cells).

[0043] (2) Aggregate induction

[0044] a. Preparation of polymer induction medium: Add ascorbic acid, L-glutamine, penicillin and streptomycin to α-MEM medium. The concentration of ascorbic acid is 50 μg / mL, the concentration of L-glutamine is 2 mM, the concentration of penicillin is 100 U / mL, and the concentration of streptomycin is 100 g / mL. The medium is renewed every 3 days;

[0045] b. Cells with a fusion rate of 80% (unsorted dental mesenchymal stem cells, PDGFRA + Dental mesenchymal stem cells and PDGFRA - Dental mesenchymal stem cells) were taken out of the incubator, the culture medium in the well plate was discarded, and 1 mL of sterile PBS was slowly added along the side wall for washing, and this was repeated twice;

[0046] c. After discarding PBS, add 2 mL of polymer induction medium and culture in an incubator (37°C, 5% CO2);

[0047] d. After 3 days, replace the aggregate induction medium and continue culturing in the incubator for 3 days;

[0048] e. Replace the culture medium in the dish with PBS, and then observe the polymer morphology with the naked eye and under a microscope;

[0049] f. After 7 days, cell clusters (aggregates) with white membranous structures were formed and were collected from the culture plate using a cell scraper.

[0050] (3) Rat periodontal bone defect modeling

[0051] a. Sprague-Dawley rats were anesthetized with 3% isoflurane using an anesthesia machine (RWD, China), and then the right mandible was exposed;

[0052] b. A periodontal bone defect with a size of 3 mm × 2 mm × 1 mm (width, height, depth) was created on the buccal side of the first molar using a 0.8 mm diameter ball drill;

[0053] c. After the defect was established, the three polymers were implanted respectively, and the defect without polymer implantation was used as a blank control;

[0054] d. Six weeks after surgery, the animals were euthanized by carbon dioxide, and the right mandibles were collected into 4 ml EP tubes and fixed with 4% PFA for 24 h;

[0055] e. Replace 4% PFA with PBS and wash three times on a shaker for the next analysis.

[0056] (4) Micro-CT analysis

[0057] a. The samples were prepared for micro-computed tomography using a PerkinElmer™ Quantum GX2 micro-computed tomography scanner (PerkinElmer, USA) at the Air Force Medical University Animal Center. Scanning parameters included an energy setting of 90 kV and a current of 80 μA, with a resolution of 18 μm;

[0058] b. The acquired data were processed for 3D reconstruction using VG Studio MAX software (version 2023.2.1) (Volume Graphics, Germany);

[0059] c. Calculate the ratio of trabecular volume to bone tissue volume (BV / TV) and quantitatively analyze bone regeneration.

[0060] Figure 2 This is the Micro-CT analysis result of periodontal bone tissue regeneration in rats; Figure 2 A is the blank control group, unsorted group, PDGFRA + , PDGFRA - Micro-CT and three-dimensional reconstruction images of the samples obtained 6 weeks after dental stem cell aggregates were implanted into periodontal bone defects in rats; Figure 2 B is the statistical chart of the bone volume fraction BV / TV (%) of each group. Figure 2 It can be seen that the implantation of unsorted DFSC aggregates promoted periodontal bone regeneration, while PDGFRA - DFSC aggregate implantation has limited effect on periodontal bone repair; PDGFRA + The periodontal bone defects in the DFSC polymer implant group showed significant regeneration, almost restoring the original bone volume in the defect area.

[0061] (5) Histological analysis

[0062] a. The mandibles of SD rats were decalcified in 17% ethylenediaminetetraacetic acid (EDTA) for 4 weeks;

[0063] b. The completely decalcified samples were dehydrated in a series of ethanol and xylene;

[0064] c. Paraffin-embedded mandibular tissue;

[0065] d. Prepare 5 μm thick paraffin tissue sections using a microtome (Leica, USA);

[0066] e. Hematoxylin and eosin (H&E) staining was performed as previously described.

[0067] Figure 3 H&E staining of periodontal bone defect area (scale bar = 250 μm). Figure 3It can be seen that H&E staining histological analysis confirmed that compared with PDGFRA - DFSC group PDGFRA + DFSC aggregates had superior bone healing ability after implantation, with newly formed trabeculae replacing the defect area, while the blank control group had almost no bone tissue formed and fibrous tissue was present.

[0068] (6) Immunofluorescence staining analysis

[0069] a. Completely decalcified rat mandibular tissue was dehydrated in 30% sucrose solution (Sigma-Aldrich, USA);

[0070] After bOCT embedding (4583, Sakura Finetek, USA) of tissues, 10-μm-thick frozen sections were obtained in a cryomicrotome (Leica, Germany);

[0071] c. Treat with 0.3% Triton X-100 (X100PC, Sigma-Aldrich, USA) for 15 minutes at room temperature to permeabilize;

[0072] d. Blocked with goat serum (AR0009, Boster, China) for 30 min;

[0073] e. The sections were then incubated with the following primary antibodies: RUNX2 (sc-390715, Santa Cruz Biotechnology, USA; dilution 1:100), CD31 (FAB3628G, R&D Systems, USA; dilution 1:100), EMCN (DF13357, Affinity, China; dilution 1:100), rtPDGFBB (ab21234, Abcam, UK; 1:100) and hVEGFA (ab9570, Abcam, UK; 1:100) at 4°C overnight;

[0074] f. Staining with fluorescently labeled secondary antibodies for 1 hour at 37°C, followed by staining with DAPI for 10 minutes;

[0075] g. Fluorescence images were captured using a confocal laser scanning microscope (CLSM, Nikon, Japan), and the images were viewed and analyzed using NIS-Elements Viewer4.50 software;

[0076] h. Image J statistical analysis of fluorescence intensity.

[0077] Figure 4 This is an immunofluorescence staining image of the periodontal bone defect area; Figure 4A, colocalization of RUNX2 with CD31 and EMCN, as well as rtPDGFBB or hVEGFA in the regenerating bone region, scale bar = 50 μm; Figure 4 B is the quantitative analysis of the fold change of mean fluorescence intensity of RUNX2; Figure 4 C is CD31 + EMCN + Quantitative analysis of vascular percentage (%); Figure 4 D is the quantitative analysis of the fold change of the mean fluorescence intensity of rtPDGFBB; E is the quantitative analysis of the fold change of the mean fluorescence intensity of hVEGFA; n = 3 per group; *P < 0.05; **P < 0.01; ***P < 0.001. Figure 4 It can be seen that the osteogenic marker RUNX2 co-localizes with CD31 and EMCN in the vicinity of new bone and blood vessels, indicating that angiogenesis and osteogenesis are coupled. + The expression of RUNX2 in the DFSC aggregate implantation group was significantly upregulated, and CD31 + EMCN + The percentage of blood vessels increased, which showed that compared with the unsorted group and PDGFRA - Compared with the DFSC group, PDGFRA + DFSC aggregates can more effectively promote angiogenesis-osteogenesis coupling. In addition, immunofluorescence staining of recipient rat PDGFBB (rtPDGFBB) and donor human VEGFA (hVEGFA) showed that the implanted PDGFRA + DFSC aggregates may respond to PDGFBB secreted by host endothelial cells and secrete VEGFA to promote angiogenesis, thereby forming an intercellular feedback to promote rapid regeneration of periodontal bone tissue.

[0078] In summary, the PDGFRA provided in the embodiments of the present application + The preparation method of dental mesenchymal stem cell aggregates has simple steps, convenient operation, low equipment cost, short preparation cycle, and easy industrialization; in addition, the PDGFRA prepared in the embodiment of the present application + Dental mesenchymal stem cell aggregates have good ability to promote periodontal bone tissue regeneration and H-type angiogenesis, providing a feasible strategy for the regenerative treatment of bone tissue damage and degenerative lesions, and have more clinical application prospects.

[0079] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

[0080] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A PDGFRA + A method for preparing dental mesenchymal stem cell aggregates, characterized in that: include: The isolated dental mesenchymal stem cells were labeled with magnetic beads and antibodies, and PDGFRA was sorted. + Dental mesenchymal stem cells; The PDGFRA + Dental mesenchymal stem cells were induced and cultured to obtain PDGFRA + Dental mesenchymal stem cell aggregates; Wherein, the polymer induction medium is composed of the following components: α-MEM medium, ascorbic acid, L-glutamine, penicillin and streptomycin.

2. The PDGFRA according to claim 1 + A method for preparing dental mesenchymal stem cell aggregates, characterized in that: The polymer induction medium is prepared according to the following steps: Ascorbic acid, L-glutamine, penicillin and streptomycin were added to α-MEM medium. The concentration of ascorbic acid was 0-100 μg / mL, the concentration of L-glutamine was 0.5-10 mM, the concentration of penicillin was 100 U / mL, and the concentration of streptomycin was 100 g / mL.

3. The PDGFRA according to claim 2 + A method for preparing dental mesenchymal stem cell aggregates, characterized in that: The polymer induction medium is prepared according to the following steps: Ascorbic acid, L-glutamine, penicillin and streptomycin were added to α-MEM medium. The concentrations of ascorbic acid, L-glutamine, penicillin, and streptomycin were 50 μg / mL, 2 mM, 100 U / mL, and 100 U / mL, respectively.

4. The PDGFRA according to claim 1 + A method for preparing dental mesenchymal stem cell aggregates, characterized in that: The separated dental mesenchymal stem cells are labeled with magnetic beads-antibodies and PDGFRA is sorted out. + The steps of dental mesenchymal stem cells include: The dental mesenchymal stem cells separated and subcultured were then treated with enzyme digestion to obtain a single cell suspension; The single cell suspension is incubated with a biotin-labeled CD140a monoclonal antibody, the resulting cells are combined with positive selection beads, washed, and placed in a magnetic field to sort out PDGFRA + Dental mesenchymal stem cells.

5. The PDGFRA according to claim 4 + A method for preparing dental mesenchymal stem cell aggregates, characterized in that: The single cell suspension was incubated with 2uL of biotin-labeled CD140a monoclonal antibody at room temperature for 10 minutes, and the obtained cells were combined with positive selection beads, washed with 3% FBS-PBS separation buffer, and placed in a magnetic field to sort out PDGFRA + Dental mesenchymal stem cells.

6. The PDGFRA according to claim 1 + A method for preparing dental mesenchymal stem cell aggregates, characterized in that: The induction culture conditions are: culture at 37°C and 5% CO2 for 7-14 days.

7. A PDGFRA + The dental mesenchymal stem cell aggregate is characterized by: The PDGFRA + The dental mesenchymal stem cell aggregate is a PDGFRA according to any one of claims 1 to 6 + The dental mesenchymal stem cell aggregates are prepared by the preparation method.

8. A PDGFRA according to claim 7 + Application of dental mesenchymal stem cell aggregates in the preparation of drugs for the treatment of periodontal tissue defects.

9. A drug for treating periodontal tissue defects, characterized in that: The drug comprises the PDGFRA according to claim 7 + Dental mesenchymal stem cell aggregates, and pharmaceutically acceptable excipients and carriers.

Images