Preparation for enhancing cell regeneration treatment capacity and application thereof

Abstract

The invention provides a preparation for enhancing cell regeneration treatment capacity and application thereof, and belongs to the technical field of biology. According to the preparation, the multiplication capacity and osteogenic differentiation capacity of the human dental pulp stem cells are effectively improved through the synergistic cooperation of the human platelet-derived growth factor BB, the umbilical cord mesenchymal stem cell exosome, the angelica polysaccharide, the platelet lysate and the alpha-MEM basic culture medium component, so that the application effect of the preparation in dental pulp regeneration treatment is enhanced. In the preparation process, the extraction method of the umbilical cord mesenchymal stem cell exosome and the angelica polysaccharide is finely optimized, so that the activity and the purity of the exosome and the angelica polysaccharide are ensured. Secondly, the preparation provided by the invention not only improves the regeneration capacity of the dental pulp stem cells, but also provides a reliable and safe alternative scheme, and overcomes the defects in the existing culture medium system.

Description

Technical Field

[0001] The invention belongs to the field of biological technology, and in particular relates to a preparation for enhancing cell regeneration therapeutic ability and application thereof. Background Art

[0002] Dental pulp stem cells (DPSCs) are adult stem cells derived from dental pulp tissue. They have multidirectional differentiation potential and can differentiate into multiple cell types such as odontoblasts, osteoblasts, chondrocytes and neural cells. Due to their high differentiation potential and regenerative ability, DPSCs have shown great application prospects in dental restoration and regenerative medicine, especially in the fields of dental pulp regeneration, dentin repair and neural tissue repair. However, the dental pulp regeneration treatment effect of DPSCs is limited by the influence of in vitro culture, and further optimization of drugs and technical means is urgently needed to enhance its regenerative ability and clinical application value.

[0003] At present, the in vitro culture of DPSCs usually relies on traditional culture medium systems, such as DMEM or α-MEM-based basal medium supplemented with fetal bovine serum (FBS) and a small amount of additives. Although this type of culture medium can maintain the basic survival of cells, it has significant deficiencies in promoting the self-renewal and differentiation ability of DPSCs. On the other hand, the composition of fetal bovine serum is complex and there are batch differences, which not only affects the consistency of culture, but also may induce potential risks of immune response or pathogen contamination. Therefore, in view of the shortcomings of the existing technology, it is of great significance to develop a culture medium that can comprehensively enhance the regenerative treatment ability of DPSCs. Summary of the invention

[0004] The present invention provides a preparation for enhancing cell regeneration therapeutic ability and its application, thereby enhancing the dental pulp regeneration therapeutic ability of dental pulp stem cells by effectively enhancing the proliferation and osteogenesis ability of dental pulp stem cells.

[0005] In order to achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a preparation for enhancing cell regeneration therapeutic ability, the preparation comprising the following ingredients:

[0007] 50-150μg / ml human platelet-derived growth factor BB, 50-150μg / ml umbilical cord mesenchymal stem cell exosomes, 50-150μg / ml Angelica polysaccharide, 10% platelet lysate and α-MEM basal medium.

[0008] Preferably, the enhanced cell regeneration ability is to enhance the dental pulp regeneration treatment ability of human dental pulp stem cells, and the dental pulp regeneration treatment ability of human dental pulp stem cells includes the proliferation ability and osteogenic differentiation ability of human dental pulp stem cells.

[0009] Preferably, the method for preparing umbilical cord mesenchymal stem cell exosomes comprises the following steps:

[0010] (1) The umbilical cord mesenchymal stem cells at passage 3 were cultured in DMEM medium for 48 hours, and the culture supernatant was collected;

[0011] (2) Centrifuge at 300 g for 10 minutes and collect supernatant 1;

[0012] (3) Centrifuge at 2000 g for 20 min and collect supernatant 2;

[0013] (4) Centrifuge at 10,000 g for 30 minutes and collect the supernatant;

[0014] (5) Centrifugation at 100,000 g for 1 h to precipitate exosomes, and the exosome precipitate was collected;

[0015] (6) After resuspending with PBS, the mixture was centrifuged at 100,000 g for 1 hour to collect the exosome precipitate to obtain umbilical cord mesenchymal stem cell exosomes.

[0016] Preferably, the preparation method of Angelica polysaccharide comprises the following steps:

[0017] (1) Wash the Chinese Angelica sinensis, dry it, grind it into fine powder, and pass it through an 80-mesh sieve for later use;

[0018] (2) Add angelica powder and double distilled water into a container at a solid-liquid ratio of 1:15, ultrasonic frequency 40 kHz, 60 ° C, extraction time 30 minutes;

[0019] (3) collecting filtrate 1 by centrifugation, performing preliminary filtration using filter paper, and collecting filtrate 2;

[0020] (4) filtering filtrate 2 through a 0.45 μm microporous filter membrane and collecting filtrate 3;

[0021] (5) Add an equal volume of Sevag reagent to filtrate 3, shake vigorously, let stand to separate layers, take supernatant 1, repeat 4 times, and obtain supernatant 2;

[0022] (6) centrifuging supernatant 2 and collecting supernatant 3;

[0023] (7) passing the supernatant 3 through a 30 kDa ultrafiltration membrane and retaining the permeate;

[0024] (8) passing the permeate through a 10Kda ultrafiltration membrane and collecting the retentate;

[0025] (9) Add 3 volumes of 95% ethanol to the retentate, mix thoroughly, and let stand at 4°C overnight;

[0026] (10) centrifuging, collecting the precipitate, and vacuum freeze-drying the precipitate to obtain Angelica polysaccharide.

[0027] Preferably, the preparation consists of the following ingredients:

[0028] 50μg / ml human platelet-derived growth factor BB, 150μg / ml umbilical cord mesenchymal stem cell exosomes, 100μg / ml Angelica polysaccharide, 10% platelet lysate and α-MEM basal medium.

[0029] In a second aspect, the present invention provides a method for enhancing the proliferation and osteogenesis ability of human dental pulp stem cells, the method comprising the following steps:

[0030] (1) preparing an α-MEM enhanced medium containing 50-150 μg / ml human platelet-derived growth factor BB, 50-150 μg / ml umbilical cord mesenchymal stem cell exosomes, 50-150 μg / ml Angelica polysaccharide and 10% platelet lysate;

[0031] (2) inoculating human dental pulp stem cells into a culture dish and culturing them until the cell density reaches more than 80%;

[0032] (3) Remove the culture medium and add α-MEM enhanced culture medium for pretreatment for 6-48h;

[0033] (4) Collecting human dental pulp stem cells for subsequent expansion culture or osteogenic culture.

[0034] Preferably, the umbilical cord mesenchymal stem cell exosomes are prepared by the above-mentioned method for preparing umbilical cord mesenchymal stem cell exosomes;

[0035] The angelica polysaccharide is prepared by the above-mentioned preparation method of angelica polysaccharide.

[0036] Preferably, the concentration of human platelet-derived growth factor BB in the α-MEM enhanced medium is 50 μg / ml, the concentration of umbilical cord mesenchymal stem cell exosomes is 150 μg / ml, and the concentration of angelica polysaccharide is 100 μg / ml.

[0037] In a third aspect, the present invention provides an application of a composition in the preparation of a culture medium for enhancing the proliferation and osteogenesis ability of human dental pulp stem cells, characterized in that the composition consists of 50-150 μg / ml human platelet-derived growth factor BB, 50-150 μg / ml umbilical cord mesenchymal stem cell exosomes and 50-150 μg / ml angelica polysaccharide;

[0038] The umbilical cord mesenchymal stem cell exosomes are prepared by the above-mentioned method for preparing umbilical cord mesenchymal stem cell exosomes;

[0039] The angelica polysaccharide is prepared by the above-mentioned preparation method of angelica polysaccharide.

[0040] Preferably, the composition consists of 50 μg / ml human platelet-derived growth factor BB, 150 μg / ml umbilical cord mesenchymal stem cell exosomes and 100 μg / ml Angelica polysaccharide.

[0041] The beneficial effects of the present invention are:

[0042] The preparation provided by the present invention pre-treats dental pulp stem cells through the synergistic cooperation of human platelet-derived growth factor BB, umbilical cord mesenchymal stem cell exosomes, and angelica polysaccharide, significantly improving the proliferation ability and osteogenic differentiation ability of dental pulp stem cells, thereby providing strong support for the regeneration and repair of dental pulp, and has important clinical application value.

[0043] Secondly, unlike traditional culture media, the enhanced preparation of the present invention does not rely on complex fetal bovine serum with large batch differences, reducing the risk of immune response and pathogen contamination.

[0044] In summary, the preparation of the present invention has shown excellent effects in improving the proliferation and osteogenic differentiation ability of dental pulp stem cells, and has the advantages of high safety and simple operation. It provides a new and effective method for cell regeneration therapy and has important clinical and scientific research application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The difference in proliferation ability of dental pulp stem cells under different pretreatment time;

[0046] Figure 2 The proliferation ability of dental pulp stem cells is different after pretreatment with different preparations;

[0047] Figure 3 The osteogenic differentiation ability of dental pulp stem cells is different after pretreatment with different preparations;

[0048] Figure 4 The expression of BGLAP and RUNX2 proteins in dental pulp stem cells was different after pretreatment with different preparations. DETAILED DESCRIPTION

[0049] The optional embodiments of the present invention will be described in more detail below. Although the optional embodiments of the present invention are described, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0050] The terms used in this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0051] Example 1

[0052] Obtaining Umbilical Cord Mesenchymal Stem Cell Exosomes

[0053] (1) The umbilical cord mesenchymal stem cells at passage 3 were cultured in DMEM medium for 48 hours, and the culture supernatant was collected;

[0054] (2) Centrifuge at 300 g for 10 min, collect the supernatant and remove the cells;

[0055] (3) Centrifuge at 2000 g for 20 min, collect the supernatant, and remove cell debris;

[0056] (4) Centrifuge at 10,000 g for 30 min, collect the supernatant, and remove large particles such as microvesicles;

[0057] (5) Centrifugation at 100,000 g for 1 h to precipitate exosomes and collect the precipitate;

[0058] (6) After resuspending with PBS, the mixture was centrifuged again at 100,000 g for 1 hour, and the precipitate was collected to obtain umbilical cord mesenchymal stem cell exosomes.

[0059] Example 2

[0060] Obtaining Angelica Polysaccharides

[0061] (1) Wash the Chinese Angelica sinensis, dry it, grind it into fine powder, and pass it through an 80-mesh sieve for later use;

[0062] (2) Add angelica powder and double distilled water into a container at a solid-liquid ratio of 1:15, ultrasonic frequency 40 kHz, 60 ° C, extraction time 30 minutes;

[0063] (3) collecting filtrate 1 by centrifugation (4000 rpm, 15 minutes), performing preliminary filtration using filter paper, and collecting filtrate 2;

[0064] (4) filtering filtrate 2 through a 0.45 μm microporous filter membrane and collecting filtrate 3;

[0065] (5) Add an equal volume of Sevag reagent (chloroform: n-butanol = 4:1) to filtrate 3, shake vigorously and let stand to separate the layers, take supernatant 1, repeat 4 times again to obtain supernatant 2;

[0066] (6) Centrifuge supernatant 2 (4000 rpm, 15 min) and collect supernatant 3;

[0067] (7) The supernatant 3 was passed through a 30 kDa ultrafiltration membrane, the permeate was retained, and the filtrate was used as a comparative example;

[0068] (8) passing the permeate through a 10Kda ultrafiltration membrane and collecting the retentate;

[0069] (9) Add 3 volumes of 95% ethanol to the retentate, mix thoroughly, and let stand at 4°C overnight;

[0070] (10) Centrifugation (4000 rpm, 15 min), collecting the precipitate, and vacuum freeze-drying the precipitate to obtain Angelica polysaccharide.

[0071] Example 3

[0072] Prepare the stock solution required for configuration

[0073] (1) Add 90 mg of human platelet-derived growth factor BB to 20 ml of α-MEM medium, mix thoroughly, and filter with a 0.22 μm filter membrane to obtain a 5 mg / ml human platelet-derived growth factor BB stock solution;

[0074] (2) Add 90 mg of umbilical cord mesenchymal stem cell exosomes to 20 mL of α-MEM medium, mix thoroughly, and filter through a 0.22 μm filter membrane to sterilize to obtain a 5 mg / ml umbilical cord mesenchymal stem cell exosome stock solution;

[0075] (3) Add 90 mg of Angelica polysaccharide to 20 mL of α-MEM medium, mix thoroughly, and filter through a 0.22 μm filter membrane to sterilize to obtain a 5 mg / ml Angelica polysaccharide stock solution.

[0076] Example 4

[0077] Preparation of a preparation for enhancing the regenerative therapeutic ability of dental pulp stem cells 1

[0078] (1) On a clean bench (under sterile conditions), add 50 ml of α-MEM basal medium to a sterile container, then take 0.5 ml of human platelet-derived growth factor BB stock solution, 1.5 ml of umbilical cord mesenchymal stem cell exosome stock solution, and 1 ml of angelica polysaccharide stock solution and add them to the sterile container;

[0079] (2) Add α-MEM basal medium to make up to 90 mL and mix gently;

[0080] (3) Add 10 ml of platelet lysate and mix gently to obtain Preparation 1 for enhancing the regenerative therapeutic ability of dental pulp stem cells (enhanced Preparation 1).

[0081] Example 5

[0082] Preparation of a preparation for enhancing the regenerative therapeutic ability of dental pulp stem cells 2

[0083] (1) On a clean bench (under sterile conditions), add 50 ml of α-MEM basal medium to a sterile container, then take 1 ml of human platelet-derived growth factor BB stock solution, 0.5 ml of umbilical cord mesenchymal stem cell exosome stock solution, and 1.5 ml of Angelica polysaccharide stock solution and add them to the sterile container;

[0084] (2) Add α-MEM basal medium to make up to 90 mL and mix gently;

[0085] (3) Add 10 ml of platelet lysate and mix gently to obtain preparation 2 for enhancing the regenerative therapeutic ability of dental pulp stem cells.

[0086] Example 6

[0087] Preparation of a preparation for enhancing the regenerative therapeutic ability of dental pulp stem cells 3

[0088] (1) On a clean bench (under sterile conditions), add 50 ml of α-MEM basal medium to a sterile container, then take 1.5 ml of human platelet-derived growth factor BB stock solution, 1 ml of umbilical cord mesenchymal stem cell exosome stock solution, and 0.5 ml of Angelica polysaccharide stock solution and add them to the sterile container;

[0089] (2) Add α-MEM basal medium to make up to 90 mL and mix gently;

[0090] (3) Add 10 ml of platelet lysate and mix gently to obtain Preparation 3 for enhancing the regenerative therapeutic ability of dental pulp stem cells.

[0091] Example 7

[0092] Preparation of a preparation for enhancing the regenerative therapeutic ability of dental pulp stem cells 4

[0093] (1) On a clean bench (under sterile conditions), add 50 ml of α-MEM basal medium to a sterile container, then take 1 ml of human platelet-derived growth factor BB stock solution, 1 ml of umbilical cord mesenchymal stem cell exosome stock solution, and 1 ml of Angelica polysaccharide stock solution and add them to the sterile container;

[0094] (2) Add α-MEM basal medium to make up to 90 mL and mix gently;

[0095] (3) Add 10 ml of platelet lysate and mix gently to obtain preparation 4 for enhancing the regenerative therapeutic ability of dental pulp stem cells.

[0096] Comparative Example 1

[0097] Preparation of dental pulp stem cell culture preparation 1

[0098] (1) On a clean bench (under sterile conditions), add 50 ml of α-MEM basal medium to a sterile container, then take 1.5 ml of human platelet-derived growth factor BB stock solution and 1.5 ml of umbilical cord mesenchymal stem cell exosome stock solution and add them to the sterile container;

[0099] (2) Add α-MEM basal medium to make up to 90 mL and mix gently;

[0100] (3) Add 10 ml of platelet lysate and mix gently to obtain dental pulp stem cell culture preparation 1 (culture preparation 1).

[0101] Comparative Example 2

[0102] Preparation of dental pulp stem cell culture preparation 2

[0103] (1) On a clean bench (under sterile conditions), add 50 ml of α-MEM basal medium to a sterile container, then take 1.5 ml of human platelet-derived growth factor BB stock solution and 1.5 ml of Angelica polysaccharide and add them to the sterile container;

[0104] (2) Add α-MEM basal medium to make up to 90 mL and mix gently;

[0105] (3) Add 10 ml of platelet lysate and mix gently to obtain dental pulp stem cell culture preparation 2.

[0106] Comparative Example 3

[0107] Preparation of dental pulp stem cell culture preparation 3

[0108] (1) On a clean bench (under sterile conditions), add 50 ml of α-MEM basal medium to a sterile container, then take 1.5 ml of umbilical cord mesenchymal stem cell exosome stock solution and 1.5 ml of Angelica polysaccharide and add them to the sterile container;

[0109] (2) Add α-MEM basal medium to make up to 90 mL and mix gently;

[0110] (3) Add 10 ml of platelet lysate and mix gently to obtain dental pulp stem cell culture preparation 3.

[0111] Comparative Example 4

[0112] Preparation of dental pulp stem cell culture preparation 4

[0113] (1) On a clean bench (under sterile conditions), add 50 ml of α-MEM basal medium to a sterile container, then take 0.5 ml of human platelet-derived growth factor BB stock solution, 1.5 ml of umbilical cord mesenchymal stem cell exosome stock solution, and 1 ml of Angelica polysaccharide stock solution (Angelica polysaccharide prepared from the filtrate greater than 30 KDa) and add them to the sterile container;

[0114] (2) Add α-MEM basal medium to make up to 90 mL and mix gently;

[0115] (3) Add 10 ml of platelet lysate and mix gently to obtain dental pulp stem cell culture preparation 4.

[0116] Example 8

[0117] Detection of the difference in proliferation ability of dental pulp stem cells after pretreatment with the preparations prepared in the embodiment and the comparative example

[0118] Experiment 1: The present invention uses Example 1 and the control group to screen the optimal pretreatment time

[0119] (1) P3 dental pulp stem cells were inoculated into a culture dish and cultured until the cell density reached more than 80%;

[0120] (2) removing the culture medium, adding α-MEM basal culture medium containing only 10% platelet lysate to the control group, and adding the enhanced preparation 1 prepared in Example 4 to the treatment group;

[0121] (3) After pretreatment for 6 h, 12 h, 24 h, and 48 h, the cells were collected, prepared into cell suspensions, and inoculated into 96-well cell culture plates;

[0122] (4) After inoculation, the cells were cultured for 48 h, CCK-8 reagent was added, and the absorbance was detected after incubation for 2.5 h.

[0123] Table 1 Differences in proliferation ability of dental pulp stem cells under different pretreatment times

[0124]

[0125] It can be seen from the results that after pretreatment for 6 hours, treatment with the enhanced preparation prepared in Example 1 of the present invention for 6 hours can have a significant effect on the proliferation ability of dental pulp stem cells, but the effect is not optimal.

[0126] Further observation shows that after 12 hours of pretreatment, the proliferation ability of dental pulp stem cells is further improved compared with 6 hours, and the absorbance value increases significantly. When the pretreatment time is extended to 24 hours, the proliferation effect reaches a peak value, and the absorbance value is significantly improved compared with the 12-hour pretreatment, indicating that the enhanced preparation 1 fully exerts its effect within 24 hours and the cell proliferation ability is stimulated to the greatest extent. When the pretreatment time is extended to 48 hours, there is no significant difference in the absorbance value compared with the 24-hour group, indicating that 24 hours is sufficient to activate the full potential of cell proliferation, and further extending the time will not significantly enhance the promotion effect. Therefore, based on the above observations, the present invention selects 24 hours as the optimal pretreatment time.

[0127] Experiment 2: Detect the difference in absorbance between different treatment groups after pretreatment with enhanced preparations 1-4 and culture preparations 1-4 for 24 hours.

[0128] (1) P3 dental pulp stem cells were inoculated into a culture dish and cultured until the cell density reached more than 80%;

[0129] (2) The culture medium was removed, and the control group was added with α-MEM basal culture medium containing only 10% platelet lysate, treatment group a was added with enhanced preparation 1, treatment group b was added with enhanced preparation 2, treatment group c was added with enhanced preparation 3, treatment group d was added with enhanced preparation 4, and treatment group a was added with culture preparation 1, treatment group b was added with culture preparation 2, treatment group c was added with culture preparation 3, and treatment group d was added with culture preparation 4;

[0130] (3) After 24 h of pretreatment, cells were collected, prepared into a cell suspension, and inoculated into a 96-well cell culture plate;

[0131] (4) After inoculation, the cells were cultured for 48 h, CCK-8 reagent was added, and after incubation for 2.5 h, the absorbance at 450 nm was measured.

[0132] Table 2 Differences in proliferation ability of dental pulp stem cells after pretreatment with different preparations

[0133]

[0134]

[0135] From the experimental results, it can be seen that compared with the control group, the absorbance of all treatment groups (ad) and the comparison treatment group (ad) increased. This shows that the combination of two or three of human platelet-derived growth factor BB (PDGF-BB), human umbilical cord mesenchymal stem cell exosomes and angelica polysaccharide can significantly promote the proliferation of dental pulp stem cells, proving that these components have a certain proliferation-promoting effect under different combinations.

[0136] Further studies showed that under the same total usage conditions, the absorbance value of the three-combination treatment group (ad) was significantly higher than that of the two-combination control treatment group (ac). This indicates that when PDGF-BB, human umbilical cord mesenchymal stem cell exosomes and angelica polysaccharides are used in combination in a certain proportion, a synergistic promotion effect can be produced, significantly improving the proliferation capacity of dental pulp stem cells. This synergistic effect may be due to the fact that the three work together through different signal pathways to stimulate the expression of cell proliferation-related genes, achieving a more significant proliferation promotion effect in the pretreatment stage.

[0137] From the experimental results of the comparative treatment groups ad, it can be seen that the absorbance of treatment group a (50 μg / mL PDGF-BB, 150 μg / mL human umbilical cord mesenchymal stem cell exosomes and 100 μg / mL Angelica polysaccharide) is significantly higher than that of other combinations (treatment groups b, c and d), indicating that when the enhanced preparation is configured according to this ratio, it can exert the best synergistic effect.

[0138] In addition, by comparing the experimental results of treatment group a and control treatment group d, it was found that when angelica polysaccharide (molecular weight 10-30kDa) was replaced with angelica polysaccharide with a molecular weight greater than 30kDa, the synergistic promotion effect of PDGF-BB, human umbilical cord mesenchymal stem cell exosomes and angelica polysaccharide was significantly reduced at the same dosage and concentration. This shows that the molecular weight of angelica polysaccharide is an important factor affecting its biological function. Angelica polysaccharide with a molecular weight of 10-30kDa is more suitable for synergistic use with PDGF-BB and exosomes, and can more effectively promote the proliferation of dental pulp stem cells.

[0139] Example 9

[0140] Detection of the difference in the osteogenic ability of dental pulp stem cells after pre-treating dental pulp stem cells with the preparations prepared in the embodiment and the comparative example

[0141] (1) P3 dental pulp stem cells were seeded into a 12-well culture dish and cultured until the cell density reached more than 80%;

[0142] (2) The culture medium was removed, and the control group was added with α-MEM basal culture medium containing only 10% platelet lysate, treatment group a was added with enhanced preparation 1, treatment group b was added with enhanced preparation 2, treatment group c was added with enhanced preparation 3, treatment group d was added with enhanced preparation 4, and treatment group a was added with culture preparation 1, treatment group b was added with culture preparation 2, treatment group c was added with culture preparation 3, and treatment group d was added with culture preparation 4;

[0143] (3) After 24 h of pretreatment, the culture medium was removed, the cells were gently washed with PBS, and osteogenic differentiation induction medium (10% FBS, 50 μg / mL L-ascorbic acid and 10 mM sodium β-glycerophosphate) was added, and the cells were placed in a cell culture incubator and cultured for 21 days;

[0144] (4) After the culture was completed, the osteogenic differentiation induction medium was removed and the cells were washed three times with PBS;

[0145] (5) After adding 4% paraformaldehyde to fix the cells for 20 minutes, the paraformaldehyde was removed, and 1% Alizarin Red S staining solution was added. After incubation at 37°C for 30 minutes, the staining solution was removed;

[0146] (6) Add 10% cetylpyridinium chloride to each well, incubate at room temperature for 1 hour, and measure the OD value at 562 nm.

[0147] Table 3 Differences in osteogenic ability of dental pulp stem cells after pretreatment with different preparations

[0148]

[0149]

[0150] From Table 3, it can be seen that compared with the absorbance of the control group, the absorbance of treatment group a increased by 100.42%, treatment group b increased by 63.02%, treatment group c increased by 65.27%, and treatment group d increased by 71.13%;

[0151] The control treatment group a increased by 20.92%, the control treatment group b increased by 22.55%, the control treatment group c increased by 24.27%, and the control treatment group d increased by 29.70%;

[0152] It can be seen that the promoting effects of treatment groups ad on osteogenic differentiation are significantly higher than those of the control treatment group, among which the promoting effect of treatment group a is the most significant, indicating that the combined use of PDGF-BB, human umbilical cord mesenchymal stem cell exosomes and angelica polysaccharides can also produce a synergistic promoting effect on the osteogenic differentiation ability of dental pulp stem cells.

[0153] Example 10

[0154] Detection of differences in the expression of osteogenic proteins BGLAP (human osteocalcin) and RUNX2 (Runt-related transcription factor 2) of dental pulp stem cells after pretreatment of dental pulp stem cells with the preparations prepared in the examples and comparative examples

[0155] (1) P3 dental pulp stem cells were seeded into 6-well culture dishes and cultured until the cell density reached more than 80%;

[0156] (2) removing the culture medium, adding α-MEM basal culture medium containing only 10% platelet lysate to the control group, adding enhanced preparation 1 to treatment group a, and adding culture preparation 4 to control treatment group d;

[0157] (3) After 24 h of pretreatment, the culture medium was removed, the cells were gently washed with PBS, and osteogenic differentiation induction medium (10% FBS, 50 μg / mL L-ascorbic acid and 10 mM sodium β-glycerophosphate) was added, and the cells were placed in a cell culture incubator and cultured for 14 days;

[0158] (4) After the culture was completed, the culture medium was removed, the cells were gently washed with PBS, and RIPA lysis buffer was added. After lysis on ice for 30 minutes, the supernatant was aspirated after centrifugation;

[0159] (5) After BCA detection of protein concentration, loading buffer was added and boiled at 100°C for 5 min to obtain protein samples of different treatment groups;

[0160] (6) After electrophoresis, install the transfer clip and perform electrophoresis at 250 mA for 1.5 h;

[0161] (7) After electroporation, the membrane was removed and placed in 5% skimmed milk powder and blocked at room temperature for 1 h.

[0162] (8) Incubate with BGLAP primary antibody, RUNX2 primary antibody, and β-actin primary antibody at 4°C overnight;

[0163] (9) After recovering the primary antibody, wash the membrane three times with TBST and incubate with the secondary antibody at room temperature for 2 h;

[0164] (10) Remove the secondary antibody, wash the membrane three times with TBST, and then develop and expose it again.

[0165] from Figure 4 It can be seen from the results that compared with the control group, the protein expression levels of osteogenic proteins BGLAP and RUNX2 in treatment group a pretreated with enhancement preparation 1 were significantly upregulated, while the protein expression levels of osteogenic proteins BGLAP and RUNX2 in control treatment group d pretreated with culture preparation 4 were also improved to a certain extent compared with the control group, but the degree of increase was significantly worse than that in treatment group a.

[0166] The results show that when PDGF-BB, human umbilical cord mesenchymal stem cell exosomes and Angelica polysaccharide are mixed in a ratio of 50μg / mL: 150μg / mL: 100μg / mL for dental pulp stem cell pretreatment, they can synergistically promote the expression of BGLAP and RUNX2 proteins during dental pulp stem cell osteogenic culture. However, once Angelica polysaccharide is replaced, even at the same concentration ratio, no significant synergistic effect can be achieved.

[0167] Based on the above results, it can be seen that the preparation of PDGF-BB, human umbilical cord mesenchymal stem cell exosomes and Angelica polysaccharide provided by the present invention can effectively enhance the proliferation and osteogenic differentiation ability of dental pulp stem cells after being used for pretreatment of dental pulp stem cells.

[0168] The increase in the proliferation rate of dental pulp stem cells means that during the process of dental pulp regeneration, more dental pulp stem cells can participate in tissue repair and regeneration. These newly proliferated cells can migrate to the damaged dental pulp, replenish the cell population that is missing or damaged due to injury, and provide sufficient cell resources for dental pulp regeneration.

[0169] In terms of osteogenic differentiation, when dental pulp stem cells differentiate into osteoblasts, they can synthesize and secrete bone matrix components, such as collagen and calcium salts. These components are essential for building the hard tissue support structure around the dental pulp. In dental pulp regeneration treatment, the newly formed hard tissue can stabilize the tooth structure and prevent further damage to the tooth, while also providing a suitable physical environment for the regrowth of dental pulp tissue.

Claims

1. A preparation for enhancing cell regeneration therapeutic ability, characterized in that: The preparation consists of the following ingredients: 50-150μg / ml human platelet-derived growth factor BB, 50-150μg / ml umbilical cord mesenchymal stem cell exosomes, 50-150μg / ml Angelica polysaccharide, 10% platelet lysate and α-MEM basal medium.

2. A preparation for enhancing cell regeneration therapeutic ability according to claim 1, characterized in that: The enhanced cell regeneration ability is to enhance the dental pulp regeneration treatment ability of human dental pulp stem cells, and the dental pulp regeneration treatment ability of human dental pulp stem cells includes the proliferation ability and osteogenic differentiation ability of human dental pulp stem cells.

3. A preparation for enhancing cell regeneration therapeutic ability according to claim 2, characterized in that: The method for preparing umbilical cord mesenchymal stem cell exosomes comprises the following steps: (1) The umbilical cord mesenchymal stem cells at passage 3 were cultured in DMEM medium for 48 hours, and the culture supernatant was collected; (2) Centrifuge at 300 g for 10 minutes and collect supernatant 1; (3) Centrifuge at 2000 g for 20 min and collect supernatant 2; (4) Centrifuge at 10,000 g for 30 minutes and collect the supernatant; (5) Centrifugation at 100,000 g for 1 h to precipitate exosomes, and the exosome precipitate was collected; (6) After resuspending with PBS, the mixture was centrifuged at 100,000 g for 1 hour to collect the exosome precipitate to obtain umbilical cord mesenchymal stem cell exosomes.

4. A preparation for enhancing cell regeneration therapeutic ability according to claim 3, characterized in that: The preparation method of Angelica polysaccharide comprises the following steps: (1) Wash the Chinese Angelica sinensis, dry it, grind it into fine powder, and pass it through an 80-mesh sieve for later use; (2) Add angelica powder and double distilled water into a container at a solid-liquid ratio of 1:15, ultrasonic frequency 40 kHz, 60 ° C, extraction time 30 minutes; (3) collecting filtrate 1 by centrifugation, performing preliminary filtration using filter paper, and collecting filtrate 2; (4) filtering filtrate 2 through a 0.45 μm microporous filter membrane and collecting filtrate 3; (5) Add an equal volume of Sevag reagent to filtrate 3, shake vigorously, let stand to separate layers, take supernatant 1, repeat 4 times, and obtain supernatant 2; (6) centrifuging supernatant 2 and collecting supernatant 3; (7) passing the supernatant 3 through a 30 kDa ultrafiltration membrane and retaining the permeate; (8) passing the permeate through a 10Kda ultrafiltration membrane and collecting the retentate; (9) Add 3 volumes of 95% ethanol to the retentate, mix thoroughly, and let stand at 4°C overnight; (10) centrifuging, collecting the precipitate, and vacuum freeze-drying the precipitate to obtain Angelica polysaccharide.

5. A preparation for enhancing cell regeneration therapeutic ability according to claim 4, characterized in that: The preparation consists of the following ingredients: 50μg / ml human platelet-derived growth factor BB, 150μg / ml umbilical cord mesenchymal stem cell exosomes, 100μg / ml Angelica polysaccharide, 10% platelet lysate and α-MEM basal medium.

6. A method for enhancing the proliferation and osteogenic capacity of human dental pulp stem cells, characterized in that: The method comprises the following steps: (1) preparing an α-MEM enhanced medium containing 50-150 μg / ml human platelet-derived growth factor BB, 50-150 μg / ml umbilical cord mesenchymal stem cell exosomes, 50-150 μg / ml Angelica polysaccharide and 10% platelet lysate; (2) inoculating human dental pulp stem cells into a culture dish and culturing them until the cell density reaches more than 80%; (3) Remove the culture medium and add α-MEM enhanced culture medium for pretreatment for 6-48h; (4) Collecting human dental pulp stem cells for subsequent expansion culture or osteogenic culture.

7. A method for enhancing the proliferation and osteogenic capacity of human dental pulp stem cells according to claim 6, characterized in that: The umbilical cord mesenchymal stem cell exosomes are prepared by the method for preparing umbilical cord mesenchymal stem cell exosomes described in claim 3; The angelica polysaccharide is prepared by the preparation method of angelica polysaccharide described in claim 4.

8. The method for enhancing the proliferation and osteogenic capacity of human dental pulp stem cells according to claim 7, characterized in that: The concentration of human platelet-derived growth factor BB in the α-MEM enhanced culture medium is 50 μg / ml, the concentration of umbilical cord mesenchymal stem cell exosomes is 150 μg / ml, and the concentration of angelica polysaccharide is 100 μg / ml.

9. Use of a composition in preparing a culture medium for enhancing the proliferation and osteogenic capacity of human dental pulp stem cells, characterized in that: The composition consists of 50-150 μg / ml human platelet-derived growth factor BB, 50-150 μg / ml umbilical cord mesenchymal stem cell exosomes and 50-150 μg / ml angelica polysaccharide; The umbilical cord mesenchymal stem cell exosomes are prepared by the method for preparing umbilical cord mesenchymal stem cell exosomes described in claim 3; The angelica polysaccharide is prepared by the preparation method of angelica polysaccharide described in claim 4.

10. Use of a composition according to claim 9 in preparing a culture medium for enhancing the proliferation and osteogenic ability of human dental pulp stem cells, characterized in that: The composition consists of 50 μg / ml human platelet-derived growth factor BB, 150 μg / ml umbilical cord mesenchymal stem cell exosomes and 100 μg / ml angelica polysaccharide.

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