Application of deciduous tooth pulp stem cell membrane sheet secretions in promoting osteogenic differentiation of bone marrow mesenchymal stem cells and regeneration and repair of bone tissues

Abstract

The invention discloses application of deciduous tooth pulp stem cell membrane sheet secretions in bone marrow mesenchymal stem cell osteogenic differentiation and promotion of bone tissue regeneration and repair. According to the research, it is clear that the osteoblast-induced deciduous tooth pulp stem cell sheet (SS-E) has the inflammation regulation effect on macrophages, the SS-E promotes bone marrow mesenchymal stem cell osteoblast differentiation by regulating the macrophage inflammation microenvironment, and bone defect regeneration and repair are promoted through the inflammation microenvironment; it is clear that SS-E promotes rat mandible defect repair by inhibiting inflammation. The invention further provides a culture medium for promoting osteogenic differentiation of the mesenchymal stem cells and a preparation method of the culture medium. The milk tooth pulp stem cell membrane secreta concentrated essence can be efficiently utilized, stem cells in the milk tooth period can be possibly stored in advance in the future, the milk tooth pulp stem cell membrane secreta concentrated essence is used for repairing bone defects caused by tumors, trauma and the like, inflammation at the bone defects can be effectively reduced, and regeneration of bone tissue can be promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of deciduous tooth pulp stem cell membrane sheet secretion in osteogenic differentiation of bone marrow mesenchymal stem cells and promotion of bone tissue regeneration and repair. BACKGROUND

[0002] Tumors, trauma, inflammation, etc. can cause jaw bone defects, and the incidence rate thereof ranks first among oral and maxillofacial diseases. However, the human body has no strong regeneration ability like the nerve, and the bone defects with a large volume and difficult self-healing are mainly treated by two ways in clinic: one is autologous / heterologous bone transplantation, and the other is 3D printing biological scaffold and titanium alloy implantation. The former has problems such as trauma, survival difficulty after transplantation, and rejection (heterologous bone transplantation), and the latter has problems such as complicated operation, poor biocompatibility, and sometimes secondary surgery. Therefore, ideal jaw bone regeneration and repair has always been one of the major problems to be solved by oral clinicians.

[0003] At present, a large number of scholars have found that bone immunity plays a key role in bone healing. Macrophages are key immune cells that actively participate in bone immunity in the process of bone defect repair. Based on the immunogenicity of the graft and the sensitivity of the immune system, macrophages are polarized into pro-inflammatory M1 phenotype or anti-inflammatory M2 phenotype. Most exogenous bone grafts induce M1-macrophage polarization in vivo, but prolonged M1 macrophage polarization in the early, middle and late stages of bone repair will produce an inflammatory bone microenvironment that hinders bone defect repair, which seriously hinders bone tissue healing. Therefore, in-depth exploration of new means to effectively regulate the balance of the inflammatory microenvironment in bone defects is an important topic that cannot be ignored for high-quality and rapid repair of jaw bone defects, and has very high clinical value, economic benefit and social significance.

[0004] In recent years, in bone tissue engineering, in order to overcome the limitations of immune rejection, central cell necrosis and inflammation imbalance of mesenchymal stem cells (MSC) and MSC membrane directly transplanted into bone defect, many scholars further optimize the means of bone regeneration and focus on the research of various mesenchymal stem cell exudates (MSC-E). MSC-E is concentrated from MSC supernatant after high-speed centrifugation, which is safer because of no cell structure and no immune rejection when transplanted into the host, and the high concentration of biodegradable active ingredients contained therein can directly act on the tissue damage site, significantly enhance the proliferation and migration potential of MSC, and achieve more effective tissue healing. It is worth noting that after high-density inoculation of MSC, a dense cell sheet can be formed by stimulating extracellular matrix secretion without scaffold material and enzyme digestion, which is MSC membrane, and it can secrete higher content of growth-promoting cytokines in the supernatant, and the supernatant can express higher levels of stemness genes. As one of MSCs, stem cells from human exfoliated deciduous teeths (SHEDs) not only have strong proliferation and bone regeneration ability, but also have better immune regulation ability, but there is no research to show whether SHEDs exudates have bone regeneration ability. Because whether the exudates of stem cells have the functions of stem cells themselves, there are different opinions in the field of stem cell research, although some studies have shown that SHEDs-E can significantly reduce the inflammation of temporomandibular joint chondrocytes by delivering miR100-5p, but the anti-inflammatory mechanism and the mechanism of promoting bone regeneration are not the same, and there is no research to show that it can inhibit the inflammation of macrophage RAW, so the function of SHEDs-E in promoting bone regeneration needs to be further explored. After SHEDs are induced by osteogenic mineralization solution (OM), the formation of cell membrane can be accelerated and a large amount of cytokines can be secreted. The exudates of SHEDs cell sheets (SS) (SHEDs cell sheets-derived Exudates, SS-E), that is, the exudates secreted by SHEDs cell sheets, its function has not been reported. SUMMARY

[0005] The purpose of the present application is to overcome the above-mentioned defects and deficiencies in the prior art, and to provide the application of SHEDs cell membrane exudates in the preparation of products for promoting bone tissue regeneration and repair.

[0006] The second object of the present application is to provide an application of the deciduous tooth pulp stem cell membrane sheet exudate in preparing a product for promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

[0007] The third object of the present application is to provide a method for preparing a medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

[0008] The fourth object of the present application is to provide a medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

[0009] The above objects of the present application are achieved by the following technical solutions.

[0010] The present application researches and finds that after the deciduous tooth pulp stem cell membrane sheet (SHEDscell sheets-derived Exudates, SS-E) induced by the osteogenic induction liquid (OM) is added into the M1 type macrophages induced by the bacterial lipopolysaccharide (Lipopolysaccharide, LPS) and cultured, the expression amount of the M1 type related markers is down-regulated, and the expression amount of the M2 type related markers is up-regulated, the inflammatory reaction is inhibited, and it is indicated that the SS-E plays an important role in regulating the inflammation of the macrophages. In order to further verify whether the SS-E can promote the bone tissue regeneration through the inflammatory microenvironment, the present application prepares the supernatant of the M1 type macrophages pretreated by the SS-E induced by the OM into a conditioned medium (SS-E-CM), simulates the inflammatory microenvironment, and is used for culturing the bone marrow mesenchymal stem cells (BMSCs), and it is found that the medium significantly promotes the osteogenic differentiation of the BMSCs, and the mRNA and the specific molecular mechanism of the SS-E in promoting the bone defect regeneration and repair process through the inflammatory microenvironment are explored, and it is further verified through the rat mandibular defect model that the SS-E can promote the rat mandibular defect repair through the inhibition of inflammation. The above results indicate that the SS-E has important significance in the osteogenic differentiation of the BMSCs and the bone tissue regeneration and repair through the regulation of the inflammatory microenvironment of the macrophages. In summary, the present application firstly proposes the application of the SS-E induced by the OM in the regulation of the inflammation of the macrophages, and studies the ability and the specific mechanism of the SS-E in promoting the bone tissue regeneration through the inflammatory microenvironment of the macrophages, which not only can fully utilize the concentrated essence of the MSC membrane sheet exudate and improve the anti-inflammatory ability, but also can improve the immune rejection and other defects of the MSC and the membrane sheet in the transplantation process, and promote the process of the clinical application of the SS-E.

[0011] Therefore, the present application firstly provides an application of the deciduous tooth pulp stem cell membrane sheet exudate in preparing a product for promoting the bone tissue regeneration and repair.

[0012] The present application also provides an application of the deciduous tooth pulp stem cell membrane sheet exudate in preparing a product for promoting the osteogenic differentiation of the bone marrow mesenchymal stem cells.

[0013] Further, the milk tooth dental pulp stem cell membrane sheet is obtained by osteogenic induction.

[0014] Preferably, the method for preparing the milk tooth dental pulp stem cell membrane sheet secretion comprises the following steps:

[0015] S1. osteogenic induction of milk tooth dental pulp stem cells to form a milk tooth dental pulp stem cell membrane sheet;

[0016] S2. culturing the milk tooth dental pulp stem cell membrane sheet obtained in S1 to collect the culture solution;

[0017] S3. centrifuging the culture solution obtained in S2, filtering and centrifuging the supernatant, and collecting the supernatant to obtain the milk tooth dental pulp stem cell membrane sheet secretion; the molecular weight of the milk tooth dental pulp stem cell membrane sheet secretion is greater than 3 kDa.

[0018] Preferably, the culturing in step S2 is carried out using a serum-free medium.

[0019] Preferably, the milk tooth dental pulp stem cell membrane sheet obtained in S1 is cultured using iCell primary mesenchymal stem serum-free basic medium, the culture solution is collected every 24 h, and new iCell primary mesenchymal stem serum-free basic medium is added, and the culture solution is collected for a total of 3 days.

[0020] Further, the milk tooth dental pulp stem cell membrane sheet secretion promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes bone tissue regeneration and repair by regulating the inflammatory microenvironment of macrophages.

[0021] Further, the product for promoting bone tissue regeneration and repair promotes bone tissue regeneration and repair by inhibiting macrophage inflammation.

[0022] Further, the concentration of the milk tooth dental pulp stem cell membrane sheet secretion in the product is 0.8-3.2 mg / mL.

[0023] Preferably, the concentration of the milk tooth dental pulp stem cell membrane sheet secretion is 1.6-3.2 mg / mL.

[0024] Preferably, the concentration of the milk tooth dental pulp stem cell membrane sheet secretion is 1.6 mg / mL.

[0025] Further, the product for promoting bone tissue regeneration and repair is a hydrogel loaded with milk tooth dental pulp stem cell membrane sheet secretion.

[0026] Further, the product for promoting osteogenic differentiation of bone marrow mesenchymal stem cells is an osteogenic differentiation medium for bone marrow mesenchymal stem cells.

[0027] Preferably, the culture medium is a conditioned medium; the added ingredient in the conditioned medium is culturing M1 macrophages in a medium containing osteogenic-induced deciduous dental pulp stem cell membrane sheet secretions and removing the supernatant of the cells after culturing.

[0028] The application also provides a method for preparing a medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, comprising the following steps:

[0029] S1. Adding deciduous dental pulp stem cell membrane sheet secretions to a cell culture medium, culturing M1 macrophages, removing the cell precipitate after culturing, and obtaining the supernatant;

[0030] S2. Adding the supernatant obtained in S1 to a basic medium, mixing, and then adding serum and antibiotics to prepare a medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

[0031] Further, the deciduous dental pulp stem cell membrane sheet secretions in step S1 are osteogenic-induced deciduous dental pulp stem cell membrane sheet secretions.

[0032] Preferably, the concentration of the osteogenic-induced deciduous dental pulp stem cell membrane sheet secretions added in step S1 is 0.8-3.2 mg / mL.

[0033] Preferably, the concentration of the osteogenic-induced deciduous dental pulp stem cell membrane sheet secretions added in step S1 is 1.6-3.2 mg / mL.

[0034] Preferably, the concentration of the osteogenic-induced deciduous dental pulp stem cell membrane sheet secretions added in step S1 is 1.6 mg / mL.

[0035] Further, the volume ratio of the basic medium to the supernatant in step S2 is 1:1.

[0036] Further, the basic medium in step S2 is DMEM high-sugar medium.

[0037] Further, the addition of serum and antibiotics in step S2 is adding 10% FBS and 1% antibiotics by volume fraction.

[0038] The application also provides a medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells prepared by the above method.

[0039] Compared with the prior art, the application has the following beneficial effects:

[0040] The application provides application of deciduous tooth pulp stem cell membrane sheet secretions in bone marrow mesenchymal stem cell component differentiation and promotion of bone tissue regeneration and repair, the deciduous tooth pulp stem cell membrane sheet is obtained by osteogenic induction, and the application researches that the osteogenic induced deciduous tooth pulp stem cell membrane sheet secretions (SS-E) have an inflammatory regulation effect on macrophages, the SS-E promotes osteogenic differentiation of bone marrow mesenchymal stem cells by regulating the inflammatory microenvironment of macrophages, promotes bone defect regeneration and repair through the inflammatory microenvironment, and it is clear that the SS-E promotes rat mandibular defect repair by inhibiting inflammation. Further, a culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells and a preparation method thereof are provided. The application can efficiently utilize the deciduous tooth pulp stem cell membrane sheet secretion concentrate essence, and in the future, the stem cells in the deciduous tooth period can be reserved in advance, which can be used for bone defect repair caused by tumors, trauma and the like, can effectively reduce inflammation at the bone defect site, and can promote the regeneration of bone tissue. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The extraction and phenotype identification results of cells; wherein, A is microscopic observation of SHEDs cell morphology; B is FCM detection of SHEDs surface markers; C is microscopic observation of BMSCs cell morphology; and D is FCM detection of BMSCs surface markers.

[0042] Figure 2 The inflammatory regulation ability of SS-E on macrophages; wherein, A is CCK-8 detection of the toxic effect of SS-E on RAW264.7 cells; B is FCM detection of the proportion of CD206 positive cells in RAW 264.7 cells; C is qRT-PCR detection of the mRNA level related to macrophage polarization; D is Western blot detection of the influence of SS-E on the immune regulation of RAW264.7 macrophages; and E is quantitative analysis of the expression amount of inflammatory proteins.

[0043] Figure 3 The SS-E promotes osteogenic differentiation of BMSCs through the macrophage conditioned medium; wherein, A is CCK-8 detection of the influence of SS-E-CM on the cell viability of BMSCs; B is qRT-PCR detection of the influence of SS-E-CM on the expression of osteogenic related genes of BMSCs; C is WB detection of the influence of SS-E-CM on the osteogenic differentiation of BMSCs (7d); D is quantitative WB results; E is ALP staining of SS-E-CM and BMSCs co-cultured; F is the osteogenic mineralization result of SS-E-CM and BMSCs co-cultured for 14d; and G is the semi-quantitative result of the mineralization capacity of BMSCs treated by SS-E-CM.

[0044] Figure 4Figure 1. The results of SS-E pretreatment regulating the inflammatory regulation ability of Ml macrophages; A is the cluster analysis heat map of differential genes; B is the KEGG pathway enrichment analysis; C is the GSEA analysis of TNFA signaling pathway (left), JAK-STAT signaling pathway (middle) and NF-Kappa B signaling pathway (right) diagram; D is the gene expression result of IL-10 and ARG-1 after LPS+SS-E treatment of RAW264.7.

[0045] Figure 5 Figure 2. The results of in vivo animal experiment to verify the anti-inflammatory and pro-osteogenic potential of SS-E; A is the Micro-CT imaging and quantitative analysis results of SS-E promoting the mandibular defect of rats; B is the microscopic observation of H&E staining at 6 weeks (left: 10x, scale = 600 pm; right: 100x, scale = 100 pm; "B" represents newly formed bone tissue; "D" represents the bone defect site; the "dotted line" represents the bone defect site) and the quantitative results of new bone formation; C is the microscopic observation of Masson staining at 6 weeks (scale = 100 pm, "B" represents newly formed bone tissue, "D" represents the bone defect site) and the quantitative results of new bone formation; D is the immunohistochemical staining results and quantitative results at 6 weeks. DETAILED DESCRIPTION

[0046] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] Example 1

[0049] (1) Extraction of primary teeth dental pulp stem cells SHEDs and bone marrow mesenchymal stem cells BMSCs and identification: clinical collection of retained primary teeth dental pulp tissue, extraction of SHEDs by modified enzyme digestion method, cells were cultured in a-MEM medium containing 10% fetal bovine serum (FBS) and 1% double antibody (complete culture medium) at 37°C, 5% CO2 concentration in the incubator, and observed under an inverted microscope at 7-14d. A large number of cells were observed to crawl out from the periphery of the tissue block, and their morphology was observed. At this time, the cells were arranged in a vortex around the tissue block, and the cell morphology could be polygonal or fusiform. After subculture, the cell state was stable and rapidly proliferated, and the morphology was mainly fusiform (such as Figure 1 A). Flow cytometry (such as Figure 1B) showed that SHEDs highly expressed MSCs surface markers such as CD73, CD90, CD105 and CD146, an endothelial cell-derived stem cell marker of hematopoietic system, with expression rates higher than 80%; and lowly expressed STRO-1 related MSCs surface markers, CD19, CD45 lymphocyte surface markers and CD34, an endothelial cell marker of hematopoietic system, proving that the cells were MSCs derived.

[0050] BMSCs were extracted from rat tibia and subjected to isolation and culture, as shown in Figure 1 C, under an inverted microscope, it was observed that P1 and P4 BMSCs grew well and the morphology was mainly spindle-shaped. FCM results (as shown in Figure 1 D) showed that BMSCs highly expressed MSCs surface markers such as CD29, CD44 and CD90, with expression rates higher than 99%; and lowly expressed CD45, CD34 and CD31, endothelial cell-derived stem cell markers of hematopoietic system, with expression rates lower than 2%, proving that the cells were MSCs derived.

[0051] (2) Passage of SHEDs: under a microscope, when the cell growth area reached about 80% of the culture dish, the cells could be passaged, the original culture medium was discarded, the cells were washed twice with sterile PBS buffer, 2 mL of 0.25% trypsin was added and then quickly placed in a culture box for digestion for 2 min. Under a microscope, it was observed that the cells were basically detached from the bottom of the culture dish and suspended in the culture medium. When the cell suspension was added to slightly more than 2 mL of α-MEM culture medium to stop the digestion, the culture medium containing the cells was transferred to a 15 mL centrifuge tube, centrifuged at a speed of 1000 rpm for 5 min, the supernatant was discarded and the cell precipitate was retained. Then, 2 mL of fresh complete culture medium was added, the cell suspension was resuspended by repeatedly blowing with a 1 mL pipette gun, and the cell suspension was uniformly transferred to two new culture bottles. Then, 3 mL of complete culture medium was added to each culture bottle, and the culture bottles were placed in a culture box at 37°C and 5% CO2 concentration for culture. P3-P6 cells growing well were selected for experiments.

[0052] Example 2

[0053] 1. Preparation of SS-E culture medium

[0054] P3-P6 SHEDs obtained from Example 1 were added to complete medium and placed in an incubator for culture, and the medium was changed every 1-2 days. When the cell growth area reached 70-80% of the bottom of the culture bottle, the original culture medium was discarded, and the cells were washed twice with sterile PBS buffer. Fresh osteogenic induction liquid (OM) was prepared: DMEM medium containing 1% double antibody, 10% FBS, 50 μg / mL vitamin C, 10 mM β-glycerophosphate sodium, and 10 mM dexamethasone. OM was added to the culture medium to induce the formation of SHEDs cell membrane sheets (SS) for about 10 days. The original culture medium was discarded, and the cells were washed twice with sterile PBS buffer. iCell primary mesenchymal stem serum-free basal medium (Shanghai Sibionan Biotech Co., Ltd.) was used for culture. The culture medium was collected every 24 hours using a centrifuge tube, and fresh iCell primary mesenchymal stem serum-free basal medium was added. The culture medium was collected for 3 days, centrifuged at 1000 rpm for 5 min, and the supernatant was collected. The supernatant was filtered through a 0.25 μm filter, and the concentrated supernatant above the filter tube was collected by centrifugation at 5000 x g for 1 h using a 15 mL, 3 kDa ultrafiltration centrifuge tube. The concentrated supernatant was obtained as SS-E, and the protein concentration in SS-E was detected using a BCA protein quantification kit according to the instructions. The remaining SS-E was stored in a -80°C freezer for subsequent experiments. The SS-E can be diluted to the desired concentration with sterile PBS buffer before the experiment.

[0055] 2. SS-E inhibits LPS-induced inflammatory state of macrophages

[0056] The cells were divided into a Control group (DMEM containing 10% FBS and 1% double antibody) and an SS-E group (DMEM containing 10% FBS and 1% double antibody with 0.8, 1.6, and 3.2 mg / mL SS-E, respectively) for RAW264.7 cell proliferation detection. When the RAW264.7 cells grew to 70-80% of the bottom of the culture bottle, the original culture medium was discarded, and the cells were washed twice with sterile PBS buffer. Four mL of complete medium containing 100 ng / mL LPS was added and cultured for 1 day. Under a microscope, the cells grew out obvious tentacles, indicating the formation of pro-inflammatory macrophages, which were M1 type macrophages at this time. Then the culture medium was removed, and 4 mL of complete medium containing 0, 0.8, 1.6, and 3.2 mg / mL SS-E, respectively, was added. After 1 day of culture, the expression of macrophage inflammation-related genes was detected. The groups were LPS (0 mg / mL SS-E) and LPS+SS-E (0.8, 1.6, and 3.2 mg / mL SS-E). A better anti-inflammatory concentration of the LPS+SS-E group was selected by RAW264.7 cell proliferation detection results and macrophage-related gene expression. The expression of macrophage inflammation-related proteins was detected compared with the LPS group.

[0057] RAW264.7 were added with different concentrations (0.8, 1.6, 3.2 mg / mL) of SS-E, and the CCK-8 results (as shown in Figure 2 A) showed that the proliferation ability of RAW264.7 was not inhibited by SS-E at each concentration, and then SS-E was added to the M1 macrophages induced by LPS for culture, and the qRT-PCR results (as shown in Figure 2 C) showed that SS-E at a concentration of 1.6 mg / mL had a better effect on inhibiting inflammation, so 1.6 mg / mL of SS-E was used for subsequent studies. The FCM results (as shown in Figure 2 B) and WB results and the results of quantitative analysis of inflammatory protein expression (as shown in Figure 2 D, E) showed that after adding SS-E, the expression of anti-inflammatory related genes and proteins was high, and the expression of pro-inflammatory related genes and proteins was low. The above results showed that SS-E could regulate the inflammatory microenvironment of LPS-induced macrophages.

[0058] Example 3

[0059] After screening a better anti-inflammatory concentration of SS-E according to Example 2, 4 mL of complete medium containing SS-E at a concentration of 0 mg / mL or 1.6 mg / mL was added to M1 macrophages, and after 1 d of culture, 2 mL of DMEM medium without FBS was used for culture for 6 h, and the supernatant was collected by centrifugation at 1000 rpm for 5 min to remove the cell precipitate and collect the supernatant above. Now use or place in the -80°C refrigerator for subsequent experiments.

[0060] BMSCs cell proliferation detection: The supernatant of the M1 macrophages treated with SS-E was collected to prepare the conditioned medium for culturing BMSCs. DMEM complete medium was prepared as the Control group; the supernatant: DMEM was uniformly mixed at a volume ratio of 1:1, and 10% FBS and 1% double-antibody were added as the conditioned medium, which was divided into the CM group (without SS-E) and the SS-E-CM group (with 1.6 mg / mL of SS-E).

[0061] BMSCs osteogenesis-related experiment detection: OM medium alone was prepared and recorded as the OM group; 50 μg / mL of vitamin C, 10 mM of β-glycerophosphate sodium and 10 mM of dexamethasone were added to the above-mentioned corresponding CM and SS-E-CM, and recorded as the corresponding CM group and SS-E-CM group for culturing BMSCs. BMSCs osteogenesis-related gene and protein detection, BMSCs ALP activity detection, and ARS qualitative and quantitative detection were performed.

[0062] The results are as follows:

[0063] 1, 3, 5d after observation CCK-8 proliferation results Figure 3 A) shows that SS-E-CM promotes BMSCs proliferation at 5d. qRT-PCR results (as shown in Figure 3 B) show that at 4d, the SS-E-CM group has high expression of ALP, Runx-2 and BMP-2 gene levels compared with the CM group, and the OM group has low expression of ALP, BMP-2 gene levels. At 7d, the OM group and the SS-E-CM group have high expression of ALP, BMP-2, Runx-2 gene levels compared with the CM group. WB qualitative results (as shown in Figure 3 C, 3D) show that at 7d, the OM group and the SS-E-CM group have high expression of ALP, BMP-2, Runx-2 protein levels compared with the CM group. ALP staining results (as shown in Figure 3 E) shows that at 4d and 7d, the ALP staining of the SS-E-CM group is deeper than that of the CM group and the OM group. ARS staining results (as shown in Figure 3 F) shows that at 14d, the SS-E-CM group has more mineralized nodules than the CM group and the OM group, and the staining is deeper. Semi-quantitative results (as shown in Figure 3 G) shows that the mineralization amount of the SS-E-CM group is significantly higher than that of the CM group and the OM group. The above results show that the SS-E-CM medium significantly promotes the osteogenic differentiation of BMSCs, suggesting that SS-E plays an important role in the osteogenic differentiation of BMSCs by regulating the inflammatory microenvironment of macrophages

[0064] Example 4

[0065] Mechanism of SS-E pretreatment regulating the inflammatory regulation ability of M1 macrophages

[0066] RAW 264.7 was inoculated in a 6-well plate at a cell density of 1.0 x 10 5 / well, and was cultured in a 37℃, CO2 cell incubator. When the cell density reached about 70-80% of the well plate, the original culture medium was discarded, 2mL of complete culture medium containing 100ng / mL of LPS was added to each well, and RAW 264.7 was induced into M1 macrophages after 24h. The culture medium was discarded, and the cells were washed twice with sterile PBS buffer. 2mL of conditioned medium was added to each well and divided into two groups: 2mL of complete culture medium was added to the control group (Control group); 2mL of 1.6mg / mL SS-E complete culture medium was added to the LPS+SS-E group (experimental group). Each group had three replicate wells. After 24h, the culture medium was discarded, the cells were washed twice with PBS buffer, and total RNA was obtained for mRNA sequencing.

[0067] The RNA of M1 macrophages treated by SS-E was extracted, and the mRNA with higher content and significant differential expression was screened by high-throughput sequencing to determine the key mRNA. The gene expression profile data was obtained, and the KEGG pathway enrichment analysis of the differentially expressed mRNA was performed. It was found that the differentially expressed mRNA was mainly distributed in the basic signal pathways related to inflammation regulation, such as TNF signal pathway and JAK-STAT signal pathway.

[0068] The results show that the top 5 differentials contain TNF signal pathway and JAK-STAT signal pathway, and the differential genes enriched in the two pathways all contain IL-10 and Arg-1, indicating that SS-E may inhibit the inflammatory state of macrophages by targeting Arg-1 / IL-10 (A-C); qRT-PCR detection results (D) show that compared with the control group, the LPS+SS-E group up-regulates the expression levels of IL-10 and ARG-1 genes in RAW264.7, and the differences are statistically significant (p<0.05). The results are consistent with the mRNA sequencing data, indicating that the high-throughput sequencing results are reliable. Figure 4 Figure 4 D) show that compared with the control group, the LPS+SS-E group up-regulates the expression levels of IL-10 and ARG-1 genes in RAW264.7, and the differences are statistically significant (p<0.05). The results are consistent with the mRNA sequencing data, indicating that the high-throughput sequencing results are reliable.

[0069] Example 5

[0070] The mandibular molar area bone defect model of SD rats was established, and SS-E was combined with Gel-MA gel scaffold to implant into the bone defect site. The effects of SS-E on inflammatory infiltration and bone tissue generation in the bone defect site were explored by Micro-CT and tissue staining. The specific methods are as follows:

[0071] After ultraviolet disinfection of the clean bench, the solution (avoid light) was prepared according to the instructions (methyl methacrylate gelatin, Suzhou EFL Industrialization Company, China): 0.25% (W / V) initiator standard solution and 10% (W / V) Gel-MA solution. After mixing under light protection, the centrifuge tube was treated with repeated sterilization for 3 times at high temperature 80℃ for 30 min and low temperature ice water mixture for 5 min. Then, the liquid was maintained at 37℃ constant water tank under light protection, and then transferred to the clean bench for rapid dilution with sterile PBS buffer to a concentration of 1.6 mg / mL. After mixing, the liquid was quickly transferred to a culture dish with a liquid height of about 3 mm under ultraviolet light. The irradiation area was larger than the area of the culture dish to ensure uniform light. At this time, the light-cured gel was formed. The control group was mixed with the same amount of sterile PBS. Two groups were designed for in vivo study: 1) Control group: Gel-MA group; 2) Experimental group: Gel-MA+SS-E group. The volume of 3mm×3mm×3mm was cut into cylindrical implants, which were soaked in 1.6 mg / mL SS-E solution or sterile PBS solution at 4℃ overnight for standby.​

[0072] The 6-week-old SPF SD rats were weighed and intraperitoneally anesthetized. The rats were fixed on a fixation plate, and the fur around the surgical area of the bilateral mandible was removed as much as possible to expose the skin, the surgical area was disinfected, a deep incision parallel to the lower edge of the mandibular angle was made with a surgical knife, about 1.0-1.5 cm, the muscle was separated to fully expose the surgical area under the buccal side of the mandibular body below the molar, and important nerves and blood vessels were avoided. A circular defect with a volume of 3 mm x 3 mm x 3 mm was prepared by moving the dental mobile phone under continuous cooling with normal saline. One circular defect was established on the left and right mandible, respectively. The prepared Gel-MA gel was transplanted to the defect according to the group, and the surface was fully covered with a suitable Bio-Gide absorbable biological membrane. The defect was layered and sutured with 4-0 absorbable suture. There were 6 models in each group. The rats were locally injected with drugs every 1 week after the operation, 2 times, the experimental group was locally injected with 200 μL of 1.6 mg / mL SS-E solution, and the control group was injected with the same amount of sterile PBS solution, and the drug administration lasted for 2 weeks. At 6 weeks after the operation, the rats were anesthetized by placing them in a container filled with CO2, and then the complete mandible was taken out and soaked in a centrifuge tube containing 4% paraformaldehyde, and fixed in a 4°C constant temperature box for 24 h for imaging analysis and tissue section staining.

[0073] The Micro-CT results are shown in Figure 5 A, the newly formed bone tissue in the mandibular defect of the Gel-MA+SS-E group was dense, and the osteogenesis amount was higher than that of the control group; quantitative analysis of osteogenesis showed that compared with the control group, the values of BV / TV (bone volume percentage) and BS / TV (bone surface density) of the Gel-MA+SS-E group were higher, and the difference was statistically significant (P<0.05). The above results show that SS-E can effectively enhance the osteogenic differentiation ability of osteoblasts at the bone defect site in vivo.

[0074] H&E staining (as shown in Figure 5 B) results show that a large area of bone defect region can be seen in the control group, the bone healing amount is small, and inflammatory cells gather around the circular defect; the newly formed bone of the Gel-MA+SS-E group is continuous, and the osteogenesis thickness is significantly higher than that of the control group; a large number of osteoblasts gather around the newly formed bone, and bone matrix formation exists. At 6 weeks, the Gel-MA gels in the two groups are mostly degraded, and only a small amount of blue-stained Gel-MA gel is seen on the surface of the defect. The results of quantitative analysis of osteogenesis show that the newly formed bone amount of the Gel-MA+SS-E group is significantly higher than that of the control group, and the difference is statistically significant (P<0.05).

[0075] Masson staining (as shown in Figure 5C) The results showed that the green flaky matrix of the control group was mainly distributed on the surface, the bone surface edge healed but was not continuous, and a small amount of point-like red staining blood vessels were observed in the bone degradation material area; the new bone of the Gel-MA+SS-E group was more continuous, and the bone thickness was significantly higher than that of the control group, the bone was red and green interlaced staining, indicating that the new bone had basically formed and gradually transformed into mature bone, and obvious point-like red staining blood vessels were observed in the bone degradation material area, and more mature collagen fibers were observed; the quantitative analysis results showed that the positive staining of the Gel-MA+SS-E group was higher than that of the control group, and the difference was statistically significant (P<0.05).

[0076] The results of immunohistochemical staining are as follows Figure 5 D The results showed that, compared with the control group, the iNos positive expression in the network structure of the bone tissue of the Gel-MA+SS-E group was lower, the extracellular matrix staining was shallower, the ARG-1 and OCN related marker nuclear staining was more, the extracellular matrix staining was deeper, and the positive expression was higher. This suggests that SS-E can inhibit the inflammatory microenvironment of the bone defect area and promote the repair of bone tissue, which is consistent with the results of the in vitro experiment.

Claims

1. Application of secretions from cell membranes of deciduous dental pulp stem cells in the preparation of products that promote bone tissue regeneration and repair.

2. Application of cell membrane secretions of deciduous dental pulp stem cells in the preparation of products that promote osteogenic differentiation of bone marrow mesenchymal stem cells.

3. The use according to claim 1 or 2, characterized in that: The deciduous tooth dental pulp stem cell cell sheet is obtained by osteogenic induction.

4. The use according to claim 1 or 2, characterized in that: The cell membrane secretions of the deciduous tooth dental pulp stem cells promote the osteogenic differentiation of bone marrow mesenchymal stem cells and promote the regeneration and repair of bone tissue by regulating the macrophage inflammatory microenvironment.

5. The use according to claim 1 or 2, characterized in that: The concentration of the cell membrane secretions of deciduous dental pulp stem cells in the product is 0.8 to 3.2 mg / mL.

6. The application according to claim 1, characterized in that: The product is a hydrogel loaded with secretions of cell membranes of deciduous tooth pulp stem cells.

7. The application according to claim 2, characterized in that: The product is a conditioned medium with the supernatant of M1 macrophages cultured in a medium containing secretions of cell membrane sheets of deciduous dental pulp stem cells as an added component.

8. A method for preparing a culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that: The following steps are involved: S1. Adding secretions from membrane sheets of deciduous dental pulp stem cells to cell culture medium to culture M1 macrophages, removing the cells after culture, and obtaining the supernatant; S2. Add the supernatant obtained in S1 to the basal culture medium, mix, and then add serum and antibiotics to prepare a culture medium that promotes osteogenic differentiation of bone marrow mesenchymal stem cells.

9. The method according to claim 8, characterized in that The volume ratio of the basal culture medium to the supernatant in step S2 is 1:

1.

10. The culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells prepared according to claim 8 or 9.

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