Mitochondria-enhanced dental pulp mesenchymal stem cells as well as preparation method and application thereof

Abstract

The invention is applicable to the technical field of cell engineering, and provides a mitochondria-enhanced dental pulp mesenchymal stem cell and a preparation method and application thereof, and the preparation method comprises the following steps: co-culturing platelet-rich fibrin and dental pulp mesenchymal stem cells by using a Transwell chamber to obtain the mitochondria-enhanced dental pulp mesenchymal stem cell. The platelet-rich fibrin transmits mitochondria to the dental pulp mesenchymal stem cells to improve the regeneration capacity of the dental pulp mesenchymal stem cells. According to the invention, an autologous platelet enrichment, namely platelet-rich fibrin, is used as a mitochondria donor with a respiratory function, mitochondria is continuously transferred to the dental pulp-derived mesenchymal stem cells through a cell bioengineering technology, and the mitochondria-enhanced dental pulp mesenchymal stem cells are constructed and applied to tissue damage repair; the vascular regeneration regulation and control capability of the dental pulp mesenchymal stem cells is obviously improved, and the existing mesenchymal stem cell treatment technology is obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell engineering, and in particular relates to mitochondria-enhanced dental pulp mesenchymal stem cells and a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cell (MSC) therapy has shown great potential in repairing difficult-to-heal tissue defects. These cells can be easily isolated from a variety of tissues, including bone marrow, adipose tissue, and dental pulp, and expanded in vitro. One of the important regenerative properties of MSCs is that they can secrete a variety of pro-angiogenic factors, including VEGF, FGF-2, Ang-1, MCP-1, IL-6, and PLGF, which can regulate the proliferation and migration of endothelial cells and protect them from apoptosis through local immune regulation and paracrine effects. It is worth noting that MSCs from different sources have different regenerative properties. Among them, dental pulp tissue-derived mesenchymal stem cells (DPSCs) are not only easier to obtain than bone marrow-derived mesenchymal stem cells, but also have stronger proliferation ability and higher VEGF (vascular endothelial growth factor) expression levels, so they may have more advantages in promoting angiogenesis. This view has been supported by a number of in vivo and in vitro experimental studies.

[0003] However, despite the significant application effects of MSCs in experimental studies, similar results have not been observed in clinical applications. Mitochondria, as an important site for intracellular oxidative phosphorylation, not only provide energy for cells, but also play a key role in regulating metabolic processes. Mitochondria regulate cell fate by exporting metabolites, lipids, mtDNA, reactive oxygen species and other substances. The tricarboxylic acid cycle is one of the important functions of mitochondria. It can not only produce ATP through coupled phosphorylation, but also produce metabolites such as citric acid and α-ketoglutarate (AKG), which have the function of regulating fatty acid synthesis and MSC immunomodulatory ability. Studies have shown that exogenous free mitochondria can be acquired by MSCs and change their metabolic state. In particular, exogenous mitochondria can upregulate the VEGF expression of MSCs. However, the specific mechanism of how mitochondrial function regulates DPSCs is still unclear. At the same time, there is no effective means to continuously and stably export mitochondria with respiratory function to DPSCs. Therefore, the application mode and effect of regulating the function of mesenchymal stem cells by exogenous mitochondria are still unclear. For this reason, the present invention proposes a mitochondrial enhanced dental pulp mesenchymal stem cell and its preparation method and application. Summary of the invention

[0004] The purpose of the present invention is to provide a mitochondrial enhanced dental pulp mesenchymal stem cell and a preparation method and application thereof, aiming to solve the problems raised in the above background technology.

[0005] The purpose of the present invention is achieved through the following technical solutions: A method for preparing mitochondria-enhanced dental pulp mesenchymal stem cells comprises the following steps: using a Transwell chamber to co-culture platelet-rich fibrin and dental pulp mesenchymal stem cells to obtain mitochondria-enhanced dental pulp mesenchymal stem cells.

[0006] Furthermore, the platelet-rich fibrin improves the regenerative capacity of dental pulp mesenchymal stem cells by delivering mitochondria to them; mitochondrial functions include one or more of mitochondrial dynamics, tricarboxylic acid cycle, oxidative phosphorylation, lipid metabolism, ROS production and glycolysis capacity.

[0007] Furthermore, the ratio of platelet-rich fibrin to dental pulp mesenchymal stem cells is 3 ml: 5×10 5 indivual.

[0008] Furthermore, the co-cultivation time is 24 hours.

[0009] A mitochondrial enhanced dental pulp mesenchymal stem cell prepared according to the preparation method described above.

[0010] A use of the mitochondria-enhanced dental pulp mesenchymal stem cells described above in the preparation of a drug for promoting tissue regeneration and healing of soft tissue trauma.

[0011] Furthermore, the mitochondria-enhanced dental pulp mesenchymal stem cells increase the expression of VEGF and CD31 in mouse skin wound tissue.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses an autologous platelet-rich material, platelet-rich fibrin, as a mitochondrial donor with respiratory function, and continuously delivers mitochondria to mesenchymal stem cells derived from dental pulp through cell bioengineering technology, constructs mitochondria-enhanced dental pulp mesenchymal stem cells and applies them to tissue damage repair, significantly improving the vascular regeneration regulation ability of dental pulp mesenchymal stem cells and significantly improving existing mesenchymal stem cell treatment technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The morphology of DPSCs under an optical microscope.

[0014] Figure 2The three-way differentiation identification staining of DPSCs, namely osteogenic, adipogenic and chondrogenic; wherein, a is the osteogenic differentiation identification staining of DPSCs, b is the adipogenic differentiation identification staining of DPSCs, and c is the chondrogenic differentiation identification staining of DPSCs; in the figure, ARS (Alizarin Red S), Oil Red (Oil Red O) and Alician Blue / Nuclear Fast Red (Alician Blue / Nuclear Fast Red) are used to detect osteogenic, adipogenic and chondrogenic differentiation, respectively; positive staining of Alizarin Red S indicates osteogenic differentiation, positive staining of Oil Red O indicates adipogenic differentiation, and positive staining of Alizarin Blue / Nuclear Fast Red indicates chondrogenic differentiation.

[0015] Figure 3 Transmission electron microscopy observations of PRF membranes and PRF extracts treated with or without ROT / AA; wherein, a is the TEM observation image of the untreated PRF membrane, b is the TEM observation image of the PRF membrane treated with ROT / AA, c is the TEM observation image of the untreated PRF extract, and d is the TEM observation image of the PRF extract treated with ROT / AA.

[0016] Figure 4 The fluorescence quantitative curves of the relative sustained release of PRF mitochondria treated or untreated with ROT / AA (n=6-18); wherein a is the fluorescence quantitative curve of the relative sustained release of PRF mitochondria untreated, and b is the fluorescence quantitative curve of the relative sustained release of PRF mitochondria treated with ROT / AA.

[0017] Figure 5 Transmission electron micrographs of PRF mitochondria-enhanced DPSCs pre-treated or untreated with ROT / AA; wherein, a is a transmission electron micrograph of untreated PRF mitochondria-enhanced DPSCs (scale bars are 5µm, 2µm, and 500nm, respectively), and b is a transmission electron micrograph of PRF mitochondria-enhanced DPSCs pre-treated with ROT / AA (scale bars are 5µm, 2µm, and 500nm, respectively).

[0018] Figure 6 Fluorescence microscopy images of PRF mitochondria-enhanced DPSCs treated with or without ROT / AA in advance; a is the staining of DPSCs alone in the control group (Control refers to the control group), b is the co-culture of PRF and DPSCs in the PRF-treated group, and c is the co-culture of PRF and DPSCs after pre-treatment with ROT / AA; in the figure, Mito-Tracker Green refers to the mitochondrial fluorescent probe green fluorescent dye, Dil refers to a red fluorescent dye used in the biomedical field to mark the cell membrane, and Merge refers to a single comprehensive view formed by aligning and superimposing different fluorescent labeling channels through ImageJ software to determine the transfer of exogenous mitochondria.

[0019] Figure 7 It is a quantitative detection of citrate synthase and mitochondrial / cytoplasmic citrate content in PRF mitochondrial enhanced DPSCs that were previously treated with or without ROT / AA; wherein a is a quantitative detection of citrate synthase in PRF mitochondrial enhanced DPSCs that were previously treated with or without ROT / AA, and b is a quantitative detection of mitochondrial and cytoplasmic citrate content in PRF mitochondrial enhanced DPSCs that were previously treated with or without ROT / AA.

[0020] Figure 8 Immunofluorescence staining and fluorescence semi-quantitative analysis of CD31, angiogenesis-related protein, expressed in mouse skin wound tissue (n=6); a is immunofluorescence staining of CD31, angiogenesis-related protein, expressed in mouse skin wound tissue on day 3, and b is Figure 8 The fluorescence semi-quantitative analysis in a, c is the immunofluorescence staining of the angiogenesis-related protein CD31 expressed in the skin wound tissue of mice at 7 days, and d is Figure 8 Fluorescence semi-quantitative analysis of c.

[0021] Fig. 9 Immunofluorescence staining and fluorescence semi-quantitative analysis of VEGF expressed in mouse skin wound tissue (n=5); a is immunofluorescence staining of VEGF expressed in mouse skin wound tissue on day 3, b is Fig. 9 The fluorescence semi-quantitative analysis in a, c is the immunofluorescence staining of VEGF expressed in the wound tissue of mice skin at 7 days, and d is Fig. 9 Fluorescence semi-quantitative analysis of c.

[0022] Fig.10 These are HE sections of mouse skin wounds; among them, a is the HE section of mouse skin wounds in each group (Saline, P, M, R / A+P+M, P+M) at 3 days, and b is the HE section of mouse skin wounds in each group (Saline, P, M, R / A+P+M, P+M) at 7 days. DETAILED DESCRIPTION

[0023] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0024] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0025] Example 1: Extraction, separation, identification and cultivation of DPSCs; 1) Cell acquisition and culture: 10 human teeth (non-diseased teeth extracted for treatment, patients were treated at the Stomatological Hospital of Jilin University, aged 18-25 years) were collected and immediately placed in a plastic tube containing phosphate buffered saline (PBS) (Hyclone, Logan, USA) containing 10% penicillin-streptomycin (P / S, 100x solution; Hyclone) after extraction, and placed on ice for processing within 2 h. Subsequently, the teeth were opened using a sterile diamond wheel and the pulp tissue was collected. DPSCs were obtained by in situ culture method. The specific operation was as follows: the isolated tissue was placed in a T25 tissue culture flask and cultured in α-MEM (Biosharp, Guangzhou, China) complete medium supplemented with 10% fetal bovine serum (FBS) (Procell, Wuhan, China) and 1% P / S. The culture flask was placed in a constant temperature incubator at 37°C and 5% CO2, and the culture medium was changed every three days. When the cells in the culture flask were close to 90% confluence, trypsin was used to detach the cells and subcultured at an initial density of 50%-60% confluence. After the third generation of subculture was stable, the cells were collected for testing.

[0026] 2) Identification of DPSCs; The human stem cell osteogenic, adipogenic, and chondrogenic differentiation kit (Saiye (Guangzhou) Biotechnology Co., Ltd.) was used to induce and identify the three differentiations of DPSCs. The specific operations were carried out according to the instructions provided by the manufacturer.

[0027] Results: The growth of DPSCs was observed under an optical microscope and found to be growing well, showing a spindle-shaped fibroblast morphology ( Figure 1 DPSCs were stained for osteogenic, adipogenic and chondrogenic differentiation, and the results showed that DPSCs have strong osteogenic, adipogenic and chondrogenic differentiation abilities ( Figure 2 middle ac).

[0028] Example 2: Preparation of platelet rich fibrin (PRF) and study of mitochondrial release and respiratory inhibition; 1) Preparation of platelet-rich fibrin membrane; Use disposable vacuum blood collection tubes to collect venous blood from volunteers, and then immediately centrifuge the collected blood. The centrifugation procedure is: 3000rpm, 10min. The centrifugation process must maintain a constant speed.

[0029] After centrifugation, discard the supernatant. Use medical scissors to cut off the red blood cell layer at the junction of the middle layer of light yellow gel and the lower layer of red blood cells, 1-3 mm to the side of the red blood cells.

[0030] The obtained gel is placed on multiple layers of sterile gauze, and the surface is covered with the same number of layers of sterile gauze. The gauze is squeezed and continuously pressurized so that the gel is fully dehydrated and shaped in the middle of the gauze, thereby obtaining a structurally stable platelet-rich fibrin membrane.

[0031] The prepared platelet-rich fibrin membrane was transferred to a 12-well plate, and 1 mL of α-MEM culture medium containing 10% FBS and 1% penicillin-streptomycin double antibody was added to each well.

[0032] 2) Inhibition of mitochondrial electron respiratory chain; The platelet-rich fibrin membrane prepared in 1) was divided into two groups. One group was added with 2 μM rotenone (ROT) and 2 μM antimycin A (AA) for 3 h to inhibit respiratory chain complex I and complex III, thereby blocking oxidative respiratory function. The other group was added with an equal volume of cell culture medium as a control.

[0033] 3) Observation of mitochondrial slow release; Mito-Tracker Green was used to label mitochondria in the two groups of platelet-rich fibrin membranes.

[0034] The liquid in each well of the two groups was aspirated at 2h, 4h, 6h, 8h, 24h, 3days, 7days and 10days respectively, and the fluorescence intensity at the excitation wavelength of 490nm and the absorption wavelength of 516nm was measured to evaluate the relative sustained release of mitochondria.

[0035] Treated / untreated platelet-rich fibrin membranes were prepared separately, and the membranes themselves and the extracts absorbed at 24 h were observed using a transmission electron microscope (TEM).

[0036] Results: Through transmission electron microscopy, it can be clearly seen that the PRF membranes treated with or without ROT / AA contain biologically active mitochondria ( Figure 3 a and b), and these mitochondria can slowly release into the surrounding environment ( Figure 3 c and d). Based on the fluorescence intensity measurement results, the fluorescence quantitative curve of the relative slow release of PRF mitochondria treated with or without ROT / AA was drawn. The curve shows that the release capacity of mitochondria is not affected by the respiratory inhibitor ROT / AA ( Figure 4 a and b).

[0037] Example 3: Construction of enhanced DPSCs with exogenous mitochondria; Mito-Tracker Green fluorescent probe was used to label mitochondria in PRF treated with or without ROT / AA, and Dil probe was used to label DPSCs. Next, the labeled PRF treated with or without ROT / AA was co-cultured with the labeled DPSCs using a Transwell chamber (8.0 μm pore size). During this process, DPSCs were seeded in the lower chamber of the chamber, and PRF was located in the upper chamber. The ratio of PRF to DPSCs was 3 ml PRF: 5 × 10 5 After 24 h of co-culture, DPSCs were collected for testing.

[0038] The treated DPSCs were observed under transmission electron microscopy and fluorescence microscopy.

[0039] Results: Transmission electron microscopy ( Figure 5 The results in (a and b) show that mitochondria can be seen inside the PRF mitochondria-enhanced DPSCs, whether they were treated with ROT / AA or not. This indicates that we have successfully achieved the delivery of PRF-derived mitochondria. Fluorescence microscopy ( Figure 6 This was further confirmed by the observations in Figure ac). The green fluorescence came from the PRF-derived mitochondria pre-labeled with Mito-Tracker Green, while the red signal showed the Dil-labeled DPSCs cell membrane. By comparing the ROT / AA-treated group with the untreated group, we found that there was no significant difference in fluorescence intensity and mitochondrial distribution between the two groups. This indicates that ROT / AA treatment did not affect the transfer efficiency of mitochondria. In addition, we also observed that after DPSCs obtained mitochondria, their own mitochondria underwent morphological changes, manifested as mitochondrial elongation. Therefore, the differences in the indicators measured in subsequent experiments cannot be attributed to differences in mitochondrial transfer efficiency.

[0040] Example 4: Quantitative analysis of citrate synthase (CS) and citrate acid (CA) in mitochondria-enhanced DPSCs; The cells obtained in Example 3 were subjected to quantitative analysis of citrate synthase and citric acid using a citrate synthase content detection kit and a citric acid content detection kit [Solabo Technology Co., Ltd. (Beijing)]. The specific operation was performed according to the instructions provided by the reagent manufacturer.

[0041] Results: Figure 7As shown in a and b, the citrate synthase and the citrate content in mitochondria and cytoplasm of PRF mitochondria-enhanced DPSCs, whether treated with ROT / AA or untreated, were quantitatively detected, including: DPSCs (M); PRF+DPSCs (P+M); ROT / AA+PRF+DPSCs (R / A+P+M). It can be seen that compared with PRF mitochondria-enhanced DPSCs treated with ROT / AA, untreated PRF mitochondria-enhanced DPSCs showed significant increases in all indicators. This result shows that when DPSCs obtain PRF-derived mitochondria, their citrate synthase activity is significantly increased, and the citrate content is also significantly increased, suggesting that DPSCs undergo metabolic reprogramming in this process.

[0042] Example 5: Evaluation of the effect of mitochondria-enhanced DPSCs in promoting the healing of soft tissue injuries; Before the experiment, cells were prepared according to the steps of Example 3. The entire animal experiment was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee and strictly followed the ethical principles of experimental animal welfare (SY: 2024-08-002). Wild-type male C57BL / 6 mice (6-8 weeks old) were selected as experimental subjects. Before the experiment, the mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (dose of 0.1 ml / 20 g body weight), and then fixed in a prone position. After removing the hair on the back of the mouse with a razor, it was further treated with a depilatory cream and rinsed with saline to completely expose the entire back skin of the mouse. Four circular incisions with a diameter of 5 mm were made on the back of each mouse, and the full-thickness skin was removed. The following solutions were injected into each group in equal volumes: 1) saline; 2) PRF extract (P); 3) DPSCs (M); 4) ROT / AA+PRF+DPSCs (R / A+P+M); 5) PRF+DPSCs (P+M); the wound surface was covered with a sterile dressing. The wound surface dressing was removed on days 3 and 7, and the full-thickness skin tissue of the wound was obtained. The tissue samples were fixed in 4% paraformaldehyde (PFA, pH=7.4) for 24 hours before subsequent testing.

[0043] Take the fixed tissue samples and mix them in gradient ethanol. The tissues were dehydrated in 4% paraffin, cleared with xylene, and embedded in paraffin. Subsequently, the tissue samples were sliced ​​into 3 µm sections. Antigen retrieval of the tissues was performed at 37°C using pepsin retrieval solution. After 30 min, the tissue sections were washed with TBS buffer containing 1% w / v Triton X-100. Next, nonspecific binding was blocked with immunostaining blocking solution, and then the primary antibody (rabbit anti-human VEGF and CD31) was added and incubated overnight at 4°C. The next day, after washing the sections, the tissue sections were incubated with FITC goat anti-rabbit IgG (H+L) at room temperature for 60 min and washed again. Finally, the specimens were sealed with anti-fluorescence quenching mounting solution containing DAPI, and images were acquired using a fluorescence microscope.

[0044] Take another prepared tissue section and perform hematoxylin-eosin staining according to the following steps: Hematoxylin for 10 minutes → take out, rinse with plenty of tap water → hydrochloric acid alcohol for 10 seconds → take out, rinse with plenty of tap water → ammonia water for 10 seconds → take out, rinse with plenty of tap water → observe the nuclear staining effect under the microscope → eosin for 2 minutes → take out, rinse with plenty of tap water → observe the cytoplasmic staining effect under the microscope.

[0045] Dehydration and transparent mounting: 75% ethanol for 2 min → 80% ethanol for 2 min → 95% ethanol I for 2 min → 95% ethanol II for 2 min → anhydrous ethanol I for 2 min → anhydrous ethanol II for 2 min → xylene I for 10 min → xylene II for 10 min → neutral gum mounting, air-dry. Use electron microscope to obtain images.

[0046] Results: The results of immunofluorescence staining and fluorescence semi-quantitative analysis of mouse skin wound tissue ( Figure 8 In ad and Fig. 9 In ad), we found that mitochondria-enhanced DPSCs showed stronger regulatory ability in promoting angiogenesis. Specifically, compared with the Saline control group, P group, M group, and ROT / AA+PRF+DPSCs group, the expression of VEGF and CD31 in the P+M group was significantly increased.

[0047] Observe the HE staining results of mouse skin wound tissue ( Fig.10 In a and b), we found that mitochondria-enhanced DPSCs significantly promoted wound healing. Specifically, compared with the Saline control group, P group, M group, and ROT / AA+PRF+DPSCs group, the skin wounds in the P+M group had less inflammatory cell infiltration and better healing. The results at 7 days showed that the skin wounds in the P+M group were basically healed, the epidermal stratified squamous epithelium and dermis were well healed, and the regeneration of skin appendages such as hair follicles was the same as that of the uninjured area.

[0048] Data processing and statistical analysis: ImageJ software was used to measure and obtain raw data. Before quantitative analysis of data with n≥3, Shapiro-Wilk and Browne-Forsythe tests were used to test data normality and homogeneity of variance. Tukey test was used for one-way ANOVA when the data were normally distributed and the variance was homogeneous. Otherwise, non-parametric test was used. The results were considered statistically significant when p<0.05. GraphPad Prism 10.0 was used to plot the obtained data.

[0049] Conclusion: In summary, PRF can continuously transfer mitochondria to DPSCs as a mitochondrial donor, and the mitochondrial enhanced DPSCs constructed by this can undergo metabolic reprogramming, which significantly improves the existing mesenchymal stem cell therapy technology.

[0050] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for preparing mitochondrial enhanced dental pulp mesenchymal stem cells, characterized in that: The following steps are involved: Using Transwell chamber, platelet-rich fibrin was co-cultured with dental pulp mesenchymal stem cells to obtain mitochondria-enhanced dental pulp mesenchymal stem cells.

2. The preparation method according to claim 1, characterized in that: The platelet-rich fibrin improves the regenerative capacity of dental pulp mesenchymal stem cells by delivering mitochondria to the dental pulp mesenchymal stem cells.

3. The preparation method according to claim 1, characterized in that: The ratio of platelet-rich fibrin to dental pulp mesenchymal stem cells is 3 ml: 5×10 5 indivual.

4. The preparation method according to claim 1, characterized in that: The co-cultivation time is 24 hours.

5. A mitochondrial enhanced dental pulp mesenchymal stem cell prepared according to the preparation method according to any one of claims 1 to 4.

6. Use of the mitochondria-enhanced dental pulp mesenchymal stem cells according to claim 5 in the preparation of a drug for promoting tissue regeneration and healing of soft tissue trauma.

7. The use according to claim 6, characterized in that: The mitochondria-enhanced dental pulp mesenchymal stem cells increase the expression levels of VEGF and CD31 in mouse skin wound tissue.

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