Cornu cervi pantotrichum stem cell exosome and application thereof

Abstract

The invention is suitable for the technical field of bioengineering, and provides a cornu cervi pantotrichum stem cell exosome and application thereof.The method comprises the following steps that a 3 cm section of the top end of a cornu cervi pantotrichum in the growth stage is cut out, mesenchymal layer tissue is collected and cut into tissue blocks for primary cornu cervi pantotrichum mesenchymal stem cell RMCs culture, cells are cultured through an alpha-MEM culture medium, and the cornu cervi pantotrichum stem cell exosome is obtained; purifying the primary cells through a limited dilution method to obtain stem cells; the method comprises the following steps: preparing exosome-free serum through a gradient centrifugation method in advance; and after 80% of the RMCs are fused, replacing the culture medium with an alpha-MEM complete culture medium containing 10% of the exosome-free serum, performing culture, collecting supernate, performing centrifugal operation, collecting precipitates, and performing PBS resuspension to obtain the exosome RMC-Exos. Another purpose of the embodiment of the invention is to provide the cornu cervi pantotrichum stem cell exosome which is prepared by adopting the preparation method. According to the invention, it is clear that RMC-Exos can be used as a biological material, a regeneration promoting microenvironment can be recovered, and a new way with a prospect is provided for periodontal regeneration.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a deer antler stem cell exosome and its application. Background Technology

[0002] Periodontitis causes damage to periodontal supporting tissues and is the leading cause of tooth loss in adults. Current treatments, including basic therapies and surgical procedures, cannot achieve functional regeneration of periodontal tissues. This is primarily because the periodontal inflammatory microenvironment, characterized by ischemia, oxidative stress, hypoxia, or inflammation, severely damages the natural extracellular matrix, inhibiting not only the differentiation capacity of resident mesenchymal stem cells (MSCs) but also affecting the homing ability of circulating MSCs. Therefore, there is an urgent need to find an effective method to promote periodontal tissue regeneration.

[0003] Regenerating complex structures composed of different hard and soft tissues is a formidable challenge in regenerative medicine. Periodontitis is a prime example of this challenge: it gradually destroys the unique periodontal interface formed by the tooth, alveolar bone, periodontal ligament, and cementum. This disease not only leads to tooth loss, but the inflammatory microenvironment it creates is also a significant risk factor for systemic diseases such as atherosclerosis and diabetes. Although current regenerative strategies, such as guided tissue regeneration techniques and the application of growth factors, have shown some potential, achieving predictable and complete functional restoration remains extremely difficult.

[0004] A key obstacle to successful regeneration lies in the inflammatory microenvironment. Chronic inflammation leads to a severe imbalance in the extracellular matrix, which not only impairs the differentiation capacity of endogenous mesenchymal stem cells but also affects the homing and colonization of therapeutic mesenchymal stem cells. Therefore, reconstructing a regenerative microenvironment is an indispensable prerequisite for any effective stem cell therapy. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing deer antler stem cell exosomes, aiming to solve the problems mentioned in the background art.

[0006] The present invention is implemented as follows: a method for preparing deer antler stem cell exosomes includes the following steps: A 3 cm segment from the top of the antler during the growth stage was cut off, and mesenchymal tissue was collected. The tissue was cut into tissue blocks for primary antler mesenchymal stem cell (RMC) culture. The cells were cultured in α-MEM medium, and the primary cells were purified by limiting dilution to obtain stem cells. Exosome-free serum was prepared in advance using gradient centrifugation. After the RMCs reached 80% confluence, the culture medium was replaced with α-MEM complete medium containing 10% of the serum without exosomes. After culturing for 48 hours, the supernatant was collected and centrifuged. The precipitate was collected, resuspended in PBS, and the exosomes RMC-Exos were obtained and stored at -80°C.

[0007] Another objective of this invention is to provide a deer antler stem cell exosome, which is prepared using the above-described preparation method.

[0008] Another objective of this invention is to provide an application of deer antler stem cell exosomes in the preparation of periodontal regeneration biomaterials.

[0009] This invention analyzes and studies the ability of deer antler stem cell exosomes RMC-Exos to effectively promote the proliferation and osteogenic differentiation of human gingival mesenchymal stem cells in vitro. In a rat periodontitis model, RMC-Exos significantly promotes alveolar bone regeneration, demonstrating excellent therapeutic effects. Proteomics analysis shows that RMC-Exos is uniquely and abnormally enriched with vilinkin (VTN)—an extracellular matrix protein crucial for cell adhesion and osteogenic formation—significantly exceeding the level in human MSC exosomes. Given the key role of VTN in coordinating cell-ECM adhesion, activating focal adhesion kinase signaling pathways, and regulating MSC osteogenic differentiation, it is believed that these naturally VTN-rich RMC-Exos can precisely target and reconstruct the periodontal microenvironment that promotes regeneration. Therefore, this invention clarifies that this unique, VTN-rich RMC-Exos can serve as a biomaterial capable of restoring the regenerative microenvironment, providing a promising new approach for periodontal regeneration. Attached Figure Description

[0010] Figure 1 For the cell morphology and multi-lineage differentiation results provided in the embodiments of the present invention, A represents the cell morphology of RMCs and GMSCs, scale bar = 200 μm; B represents the adipogenic differentiation of RMCs and GMSCs, scale bar = 200 μm; C represents the osteogenic differentiation of RMCs and GMSCs, scale bar = 500 μm; D represents the chondrogenic differentiation of RMCs and GMSCs, scale bar = 50 μm. Figure 2 The identification results of RMCs surface markers provided in the embodiments of the present invention; Figure 3 The results of RMC-Exos separation, extraction and identification provided in the embodiments of the present invention are as follows: A is the TEM detection of RMC-Exos and GMSC-Exos; B is the NTA detection of RMC-Exos and GMSC-Exos; C is the WB detection of surface markers of RMC-Exos and GMSC-Exos. Figure 4The biocompatibility and safety test results of RMC-Exos provided in the embodiments of the present invention are as follows: A and B represent the uptake of RMC-Exos by GMSCs, scale bar = 150 μm; C represents the healthy state of GMSCs after RMC-Exos treatment; and D represents the reduction of inflammation-induced apoptosis of GMSCs by RMC-Exos. Figure 5 The biocompatibility and safety test results of RMC-Exos provided in the embodiments of the present invention are shown in A and B, which represent the uptake of RMC-Exos by RAW264.7 cells, with a scale bar of 30 μm; CE represents the inhibition of pro-inflammatory cytokine expression in inflammatory macrophages by RMC-Exos (p<0.05). Figure 6 The biocompatibility and safety test results of RMC-Exos provided in this embodiment of the invention are as follows: AC represents the no significant differences in ALT, AST, and BUN levels in the RMC-Exos-treated group mice (p>0.05); DF represents the fluorescence imaging and statistical analysis of major organs at different time points; G represents the histological analysis of major organs. Figure 7 The osteogenic effect of RMC-Exos on human GMSCs provided in this embodiment of the invention is shown in the figures: A and B show that RMC-Exos significantly increases the expression of Ocn and Col-1 genes; C and D show that RMC-Exos promotes OCN protein expression (scale bar = 150 μm); E and F show that RMC-Exos promotes COL-1 protein expression (scale bar = 150 μm); G shows that RMC-Exos promotes the expression of both OCN and COL-1 proteins; H and I show ALP staining and statistical analysis (scale bar = 200 μm); J and K show ARS staining and statistical analysis (scale bar = 500 μm). Figure 8 To illustrate how VTN enrichment in RMC-Exos promotes osteogenic differentiation of GMSCs, the following data are presented: A: Venn analysis; B: Volcano plot; C: Heatmap; D: GO analysis; E: KEGG analysis; F: Analysis of the expression levels of the top 10 proteins; G: VTN protein expression in RMC-Exos and GMSC-Exos; H: Effect of VTN inhibitors on the expression of key osteogenic markers OCN and COL-1 proteins; IL: Effect of RMC-Exos on ALP activity and ARS-positive mineralization deposition. Figure 9 The RMC-Exos provided in this embodiment of the invention activates the FAK / MEK / ERK signaling pathway. A and B represent FAK knockdown (scale bar = 150 μm); C and D represent MEK overexpression (scale bar = 150 μm); EG represents Fak, Mek, and Erk gene expression; and H represents FAK, MEK, and ERK protein expression. Figure 10The RMC-Exos provided in this embodiment of the invention promotes osteogenic differentiation of GMSCs by activating the FAK / MEK / ERK signaling pathway. A and B are ALP staining and quantification, scale bar = 200 μm; C and D are ARS staining and quantification, scale bar = 500 μm; E is Ocn gene expression; F is Col-1 gene expression; G is OCN and COL-1 protein expression. Figure 11 The following diagram illustrates the in vivo alveolar bone regeneration promoted by RMC-Exos in this embodiment of the invention: A is a flowchart of periodontitis modeling; B and C are Micro-CT three-dimensional reconstruction and statistical analysis; D and E are HE staining and statistical analysis, scale bar = 300 μm; F is Masson staining, scale bar = 150 μm; G and H are TRAP staining and statistical analysis, scale bar = 300 μm. Figure 12 The RMC-Exos provided in this embodiment of the invention promotes alveolar bone regeneration and improves periodontitis in vivo. AD represents OCN, COL-1, and ERK immunofluorescence staining, scale bar = 150 μm; E and F represent Ocn and Col-1 gene expression; G represents OCN and COL-1 protein expression. In the attached figure, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0012] The reagents and materials used in the embodiments of this invention include: Cell culture media (including α-MEM and DMEM) and 0.25% trypsin-EDTA were purchased from Gibco (Beijing, China); fetal bovine serum and Alizarin Red S solution were purchased from OriCell (Guangzhou, China); Lipofectamine TM3000 transfection reagent was purchased from ThermoFisher Scientific (Carlsbad, USA); fluorescent dyes DiD and PKH26 were purchased from Beyotime Biotechnology (Nanjing, China) and Uni-Bio (Shanghai, China), respectively; molecular biology reagents, including TRIzol, cDNA synthesis super premix, and SYBR Green premix, were purchased from Yisheng Biotechnology (Shanghai, China); protein analysis reagents, including lysis buffer, PAGE gel preparation kit, and blocking buffer, were purchased from Ebien Biotechnology (Shanghai, China); osteogenic, chondrogenic, and adipogenic induction media were purchased from Cyagen Biosciences (Suzhou, China); BCA protein concentration assay kit, alkaline phosphatase assay kit, BCIP / NBT staining solution, and Annexin V-FITC apoptosis assay kit were purchased from Beyotime Biotechnology (Shanghai, China); Masson trichrome staining kit and tartrate-resistant acid phosphatase staining kit for histological staining were purchased from Maixin Biotechnology (Fuzhou, China) and Jiancheng Biotechnology (Nanjing, China), respectively.

[0013] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0014] Example 1: Analysis and study of deer antler mesenchymal stem cells (RMCs), exosomes (RMC-Exos), and their performance: method: 1. Cell culture, identification, and transfection (all procedures involving human tissues were approved by the Medical Ethics Committee of Jilin University Stomatological Hospital (Approval No.: SJDKQ2024007), and informed consent was obtained from all donors): Human gingival mesenchymal stem cells (GMSCs) were isolated from healthy gingival tissue, and the tissue was minced to approximately 1 mm. 3 Small pieces were placed in α-MEM medium containing 10% FBS and 1% penicillin / streptomycin and cultured in a 37°C, 5% CO2 incubator. GMSCs were purified by limiting dilution method. All experiments used third-generation cells. A 3 cm segment from the tip of a deer antler during its growth stage was harvested, and mesenchymal tissue was collected and cut into 1 mm pieces. 3 The tissue blocks were used to culture primary deer antler mesenchymal stem cells (RMCs). The cells were cultured in α-MEM medium to obtain exosomes. The primary cells were purified by limiting dilution to obtain stem cells. All experiments used third-generation cells. Flow cytometry (BD FACSCalibur) was used to detect surface markers CD73, CD90, and CD105 (positive) and CD34, CD45, and HLA-DR (negative) in GMSCs to confirm their MSC phenotype. Osteoblastic, chondrogenic, and adipogenic differentiation in RMCs and GMSCs was induced to verify their multi-lineage differentiation capacity. For gene modification, when the GMSC confluence reached 70-90%, Lipofectamine was used. TM In Opti-MEM, 3000 transfected cells with siRNA or MEK overexpression plasmids targeting FAK, following the manufacturer’s instructions. Transfection efficiency was confirmed by flow cytometry 6 hours after transfection.

[0015] 2. Laboratory animals (All animal experiments were approved by the Laboratory Animal Ethics Committee of the School of Basic Medical Sciences, Jilin University (Approval Nos.: 2024337 and 202516)): Eight-week-old male C57BL / 6J mice and male Sprague-Dawley rats (200 ± 20 g) were housed in a specific pathogen-free environment with a 12-hour light / dark cycle, controlled temperature (25 ± 2℃) and humidity (60 ± 10%), and free access to food and water. 3. Isolation and identification of exosomes: Exosome-free serum was prepared in advance by gradient centrifugation (1000g centrifugation for 5 minutes, 10000g centrifugation for 30 minutes and 100000g ultracentrifugation for 4 hours); After RMCs and GMSCs reached 80% confluence, the culture medium was replaced with α-MEM medium containing 10% of the above serum and cultured for 48 hours. The supernatant was collected and then centrifuged at 1000g for 5 minutes, 10000g for 30 minutes, and 100000g for 4 hours. The precipitate was collected, resuspended in PBS, and stored at -80℃ to obtain exosomes RMC-Exos and GMSC-Exos. Exosome morphology was observed by transmission electron microscopy (TEM), and their particle size distribution and concentration were determined by nanoparticle tracking analysis (NTA). The expression of exosome surface proteins CD63 (+), HSP70 (+), and Calnexin (-) was verified by Western blotting (WB).

[0016] 4. Proteomics analysis: The proteomic composition of RMC-Exos was analyzed by ELISA using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Protein lysates were quantified, separated by SDS-PAGE, and digested with trypsin in the gel. The resulting peptides were desalted and analyzed by LC-MS / MS. The raw data were analyzed using Spectronaut Pulsar™ 18.4 software, and the UniProt database was searched. Functional annotation of the identified proteins was performed using gene ontology and pathway analysis from the Kyoto Encyclopedia of Genes and Genomes.

[0017] 5. In vitro exosome uptake experiment: To visualize cellular uptake, RMC-Exos were labeled with the fluorescent dye PKH26, and GMSCs (2 × 10⁻⁶) were added. 4 After 24 hours of serum starvation, cells were co-incubated with PKH26-labeled RMC-Exos for 12, 24, and 48 hours, respectively. After incubation, cells were washed with PBS, fixed with 4% paraformaldehyde, and the nuclei were counterstained with DAPI. Subsequently, imaging was performed using laser scanning confocal microscopy, and quantitative analysis was performed by flow cytometry. RAW264.7 cells were co-cultured with RMC-Exos for 6, 12, and 24 hours, and the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in RMC-Exos-treated macrophages were analyzed by RT-qPCR.

[0018] 6. Cell viability and apoptosis detection: The effect of RMC-Exos on GMSC viability was assessed using the Cell Counting Kit-8 method. GMSCs were seeded in 96-well plates and co-cultured with different concentrations of RMC-Exos for 1, 3, and 5 days. Optical density was measured at 450 nm. Simultaneously, apoptosis was quantitatively analyzed at the same time points using the Annexin V-FITC / propidium iodide apoptosis detection kit, and the results were analyzed by LSCM and Fc.

[0019] 7. In vitro osteogenic induction experiment: GMSCs were cultured in osteogenic induction medium supplemented with RMC-Exos. After 14 days of culture, ALP activity was assessed by BCIP / NBT staining and quantitative colorimetric method (detection of OD 405 nm absorbance). After 21 days of culture, the deposition of calcium mineralization nodules was assessed by ARS staining. Subsequently, the dye was eluted with 10% hexadecylpyridine chloride, and the absorbance was measured at 562 nm for quantification.

[0020] 8. In vivo distribution tracking: To track the in vivo distribution of RMC-Exos, it was labeled with the near-infrared dye DiD and administered to Balb / c mice via tail vein injection. Major organs (heart, liver, spleen, lung, and kidney) were harvested at 8, 16, 24, 48, and 72 hours post-injection. In vitro imaging was performed using an in vivo imaging system, and radiometric efficiency was quantified using Living Image 4.4 software.

[0021] 9. Construction and treatment of experimental periodontitis models: A periodontitis model induced by ligation wire was established in male SD rats. After one week of acclimatization, the cervical region of the maxillary second molar was ligated with 4-0 silk thread. The control group underwent sham surgery, i.e., the silk thread was removed immediately after ligation. Rats with successful ligation were randomly divided into three groups: PBS group, GMSC-Exos group, and RMC-Exos group. Each treatment agent (2µL) was injected into the periodontal pocket by local injection once every two days for a total of seven injections. All rats were sacrificed on the 14th day after the first treatment, and tissue samples were collected.

[0022] 10. Micro-CT Analysis: After the collected maxilla was fixed, it was scanned using a high-resolution micro-computed tomography system to assess alveolar bone regeneration, reconstruct three-dimensional images, and measure the distance from the cementoenamel junction to the alveolar ridge crest using Mimics Innovation Suite software to quantify the amount of bone loss.

[0023] 11. Histological and immunofluorescence analysis: After fixation and decalcification with 10% EDTA, the maxilla was embedded in paraffin and sectioned (3 µm thick). H&E staining (morphological assessment), Masson trichrome staining (collagen deposition), and TRAP staining (osteoclast activity) were performed. For immunofluorescence analysis, the dewaxed sections underwent antigen retrieval, and were then incubated overnight at 4°C with primary antibodies against OCN and type I collagen, followed by incubation with fluorescent secondary antibodies. Images were acquired via LSCM.

[0024] 12. Western blot analysis: Total protein was extracted from cells or tissues using RIPA lysis buffer containing protease inhibitors. Protein concentration was determined by the BCA method. Equal amounts of protein were separated by SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and incubated with the primary antibody overnight at 4°C. Then it was incubated with horseradish peroxidase-labeled secondary antibody and protein bands were visualized using an enhanced chemiluminescent substrate.

[0025] 13. Real-time quantitative PCR: Total RNA was extracted using TRIzol reagent and reverse transcribed into cDNA. RT-qPCR was performed using SYBR Green premixed buffer on a quantitative PCR instrument, with GAPDH as an internal control. -ΔΔCt The relative expression level of genes can be calculated.

[0026] 14. Statistical Analysis: All quantitative data are expressed as mean ± standard error of at least three independent experiments. Statistical comparisons between two groups were performed using the two-tailed Student's t-test, and comparisons among multiple groups were performed using one-way ANOVA with the Tukey test. All analyses were performed using GraphPad Prism software, and a p-value < 0.05 was considered statistically significant.

[0027] II. Results: 1. Identification of deer antler mesenchymal stem cells (RMCs) and their exosomes (RMC-Exos): Primary RMCs and GMSCs were isolated and cultured according to methods 1 and 3. Both exhibited typical spindle-shaped, fibroblast-like morphology. Figure 1 To confirm their MSC identity, their multi-lineage differentiation potential was first assessed. ARS, ALP, Oil Red O, and Alcian Blue staining confirmed that both cell types could robustly differentiate into osteogenic, adipogenic, and chondrogenic lineages. Figure 1 (Chinese BD) Flow cytometry analysis further revealed that these cells uniformly and highly expressed the positive markers CD73, CD90, and CD105, while expressing almost no hematopoietic lineage markers CD34 and CD45, as well as HLA-DR, a major histocompatibility complex class II molecule associated with professional antigen-presenting cells. Figure 2 ); After verifying the cell components, exosomes were isolated from the conditioned medium of RMCs and GMSCs using standard ultracentrifugation. The isolated vesicles were rigorously identified, and transmission electron microscopy showed that they were homogeneous cup-shaped, double-membrane vesicles, consistent with typical exosome morphology. Figure 3 (A) Nanoparticle tracking analysis quantified the particle size distribution, and the results showed that its main peak was concentrated at an average diameter of 103.5 nm, which falls entirely within the expected exosome size range. Figure 3 (B) Western blot analysis confirmed the purity of exosomes. The results showed enrichment of classic exosome markers—tetraspan membrane protein CD63 and cytoplasmic heat shock protein HSP70—while endoplasmic reticulum protein Calnexin (a common marker of cellular contamination) was significantly absent. Figure 3 (C); In summary, the above identification results verify the identity and purity of RMCs, GMSCs and their respective exosomes.

[0028] 2. RMC-Exos exhibits excellent biocompatibility and good safety both in vitro and in vivo: According to methods 5, 6, and 8, a multifaceted safety assessment of RMC-Exos was conducted. First, it was confirmed that RMC-Exos could effectively communicate with target human cells. After incubation for 12, 24, and 48 hours, respectively, an increasing accumulation of PKH26-labeled RMC-Exos was observed around the nuclei of GMSCs over time. Figure 4 (A), which is consistent with the uptake pattern of allogeneic GMSC-Exos by GMSCs; further quantitative analysis of cell uptake of RMC-Exos by flow cytometry showed that, consistent with the fluorescence image results, the uptake of RMC-Exos continued to increase within the observed time points. Figure 4 (B); Subsequently, GMSCs were co-cultured with RMC-Exos for 1, 3, and 5 days, respectively, and apoptosis was further assessed by flow cytometry. After treatment with RMC-Exos or GMSC-Exos, more than 90% of GMSCs remained healthy. Figure 4 (C), while RMC-Exos alleviated inflammation-induced apoptosis of GMSCs ( Figure 4 (D) Furthermore, when RAW264.7 cells were co-cultured with RMC-Exos for 6, 12, and 24 hours, RMC-Exos was observed to accumulate around the nuclei of RAW264.7 cells. Figure 5 AB), RT-qPCR analysis showed ( Figure 5 In macrophages treated with RMC-Exos, the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly lower than those in the LPS-activated group, and comparable to the baseline levels in the unactivated group and the GMSC-Exos-treated group. To assess the systemic biocompatibility and potential toxicity of RMC-Exos in vivo, key serum markers of liver and kidney function were first tested, such as... Figure 6 As shown in AC, compared with the control group, there were no significant differences in ALT, AST, and BUN levels in the RMC-Exos-treated group mice (p > 0.05), indicating that no obvious hepatotoxicity or nephrotoxicity was observed. The study investigated the biodistribution of intravenously injected DiD-labeled RMC-Exos in vivo. In vitro fluorescence imaging of major organs at different time points showed that exosomes mainly accumulated in the liver and spleen. Figure 6 The quantitative analysis of total radiation efficiency (D) confirmed the above findings. Figure 6 (EF); Finally, to assess tissue compatibility at the tissue level, major organs such as the heart, liver, spleen, lungs, and kidneys were collected for histological analysis, such as... Figure 6 As shown in Figure G, no obvious pathological abnormalities, such as inflammatory cell infiltration, necrosis, or significant tissue damage, were observed in any of the examined organs; therefore, the above results indicate that systemically administered RMC-Exos exhibits excellent biocompatibility and good safety.

[0029] 3. RMC-Exos enhances the osteogenic effect of human GMSCs in vitro: To explore the therapeutic potential of RMC-Exos on GMSCs under inflammatory conditions, according to methods 5, 7, 11, 12, and 13, the expression of key osteogenic markers was detected. Based on previous reports showing that GMSC-Exos has strong osteogenic capacity, it was used as a control for comparison. RT-qPCR analysis showed that LPS stimulation significantly inhibited the gene expression of OCN and COL-1 in GMSCs. Although both GMSC-Exos and RMC-Exos could reverse this inhibition, the upregulation effect of RMC-Exos on OCN and COL-1 expression was significantly stronger than that of GMSC-Exos. Figure 7 This trend was consistently confirmed at the protein level. Immunofluorescence staining showed that the reduced OCN and COL-1 expression in the LPS group was significantly restored by RMC-Exos, and the fluorescence signal induced by RMC-Exos was significantly stronger than that induced by GMSC-Exos. Figure 7 Quantitative analysis of mean fluorescence intensity confirmed that RMC-Exos induced the highest expression of OCN and COL-1, significantly superior to the GMSC-Exos treatment group. Figure 7 Furthermore, Western blot analysis validated the above results, indicating that RMC-Exos can most effectively restore the levels of OCN and COL-1 proteins suppressed by LPS. Figure 7 In terms of function, LPS treatment significantly inhibited osteogenic differentiation of GMSCs, as evidenced by a significant decrease in ALP activity and impaired matrix mineralization in ARS staining. Figure 7 In the GMSC-Exos group, RMC-Exos effectively reversed these inhibitory effects, significantly enhanced ALP staining intensity and calcified nodule formation, and this osteogenic effect was significantly better than that of the GMSC-Exos treatment group.

[0030] 4. High expression of VTN enriched in RMC-Exos promotes osteogenic differentiation of GMSCs: To elucidate the molecular basis of the unique osteogenic effect of RMC-Exos, label-free quantitative proteomics was used according to methods 4, 7, 12, and 13 to systematically compare the protein composition of RMC-Exos and GMSC-Exos. A total of 1512 proteins were identified, of which 668 were common to both groups, and 519 and 325 proteins were specific to RMC-Exos and GMSC-Exos, respectively. Figure 8 Volcano plot analysis showed significant differences in proteomes between the two groups. Compared with GMSC-Exos, 139 proteins were significantly upregulated and 129 were significantly downregulated in RMC-Exos. Figure 8 (B) Hierarchical clustering analysis heatmaps further confirmed the existence of significant and reproducible patterns of protein expression differences between the two groups. Figure 8 (C) Through GO and KEGG pathway enrichment analysis of upregulated proteins, it was found that the functional characteristics of RMC-Exos-specific proteins were significantly enriched in cellular component items such as "extracellular region", "extracellular space" and "cytoplasm", as well as biological process items such as "cell adhesion" and "proteolysis". Figure 8 KEGG pathway analysis further reinforced this extracellular matrix association, showing a significant enrichment of key cell-matrix interaction pathways such as "ECM-receptor interaction" and "focal adhesion." Figure 8 In the middle E), mass spectrometry data identified VTN as one of the most significantly upregulated proteins in RMC-Exos. Figure 8 The results were confirmed by Western blot analysis, showing that the VTN protein level in RMC-Exos was significantly higher than that in GMSC-Exos. Figure 8 (G), key mechanism verification experiments showed that the upregulation of osteogenic key markers OCN and COL-1 protein expression by RMC-Exos could be completely blocked by VTN inhibitors (G). Figure 8 In the H group, the osteogenic effect of RMC-Exos was completely reversed after the use of VTN inhibitors: ALP activity and ARS-positive mineralization deposition both decreased to the level of the LPS-treated group. Figure 8 (IL) to prove that VTN is the core molecule mediating the functional effect of RMC-Exos.

[0031] 5. VTN-rich RMC-Exos promotes osteogenic differentiation of GMSCs by activating the FAK / MEK / ERK signaling pathway: According to methods 1, 7, 12, and 13, VTN, as an upstream ligand, can trigger the FAK / MEK / ERK signaling cascade through integrin-mediated FAK phosphorylation. To elucidate the signaling pathway mechanism by which RMC-Exos promotes osteogenic differentiation of GMSCs under inflammatory conditions, the effectiveness of the gene modification tool was first verified. Immunofluorescence and flow cytometry confirmed the successful FAK knockdown and MEK overexpression. Figure 9 (AD), and then further investigation was conducted on the FAK / MEK / ERK signaling cascade. RT-qPCR analysis showed that LPS stimulation significantly inhibited the gene expression of Fak, Mek, and Erk, while RMC-Exos treatment not only reversed this inhibition but also significantly upregulated the expression levels of the three genes. Figure 9Western blot analysis further confirmed this activation effect at the protein level, showing that the levels of FAK, MEK, and ERK proteins were correspondingly increased in the RMC-Exos treatment group. Figure 9 (H) Importantly, this pathway activation ultimately translates into functional repair: RMC-Exos significantly restored ALP activity inhibited by LPS ( Figure 10 (AB) and matrix mineralization capacity ( Figure 10 CD), while in genes ( Figure 10 (EF) and protein levels ( Figure 10 The expression of osteogenic markers OCN and COL-1 was restored on G.

[0032] 6. RMC-Exos, rich in VTN, promotes alveolar bone regeneration and improves periodontitis in vivo: According to methods 2, 9, 10, 11, 12, and 13, a rat model of ligature-induced periodontitis was established to evaluate the therapeutic effect of RMC-Exos in a clinically relevant context. Figure 11 Two weeks later, Micro-CT analysis showed that, compared with the healthy control group, the PBS treatment (ligation) group exhibited severe vertical alveolar bone resorption, with a significantly increased distance from the cementoenamel junction to the alveolar ridge crest. In contrast, local injection of RMC-Exos significantly maintained alveolar bone volume and height, restoring periodontal tissue to levels close to those of the healthy control group. This regenerative effect was significantly superior to the GMSC-Exos treatment group. Figure 11 (Bronchoalveolar bone), histological analysis further assessed tissue-level regeneration. H&E staining results validated the Micro-CT findings: the PBS group showed severe destruction of periodontal attachment structures and disordered periodontal ligament fiber arrangement; while RMC-Exos treatment restored the structurally regular periodontal ligament and maintained alveolar bone integrity. Figure 11 Masson's trichrome staining showed that the RMC-Exos group had a large amount of dense and orderly collagen fibers deposited in the periodontal ligament, similar to the control group, while the collagen matrix in the PBS group was sparse and disordered (Figure 11F). To investigate the effect on bone resorption, TRAP staining results showed that the PBS group had a large number of TRAP-positive osteoclasts infiltrating along the bone surface, while RMC-Exos treatment significantly reduced the number of osteoclasts to near the level of healthy controls, indicating that it has a strong anti-bone resorption effect. Figure 11 (Middle GH). Immunofluorescence staining showed that the expression of key osteogenic markers OCN and COL-1 in periodontal tissues of the RMC-Exos treatment group was significantly upregulated, in stark contrast to the weak expression in the PBS group. Figure 12Consistent with in vitro studies, a significant increase in ERK expression was observed in the RMC-Exos group, suggesting that the FAK / MEK / ERK pathway is activated in vivo. Figure 12 To provide molecular-level evidence, RT-qPCR and Western blot analyses were performed on the obtained periodontal tissues. RT-qPCR results showed that the expression of OCN and COL-1 genes was severely downregulated in the PBS group, while RMC-Exos treatment significantly reversed and upregulated the expression of these key osteogenic genes to near the levels of the healthy control group, showing better efficacy than GMSC-Exos. Figure 12 (DE), Western blot analysis validated this result at the protein level, indicating that RMC-Exos effectively restored the reduced OCN and COL-1 protein expression in the periodontitis model ( Figure 12 (Middle F).

[0033] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A preparation method of a Cervus elaphus L stem cell exosome, characterized in that, Comprising the following steps: A 3 cm section of the top end of the growth stage of pilose antler is cut off, mesenchymal layer tissue is collected, and the tissue is cut into tissue blocks for primary pilose antler mesenchymal stem cell (RMC) culture. The cells are cultured in α-MEM medium, and the primary cells are purified to obtain stem cells by limited dilution method; The serum without exosomes is prepared in advance by a gradient centrifugation method; After the RMCs reach 80% confluence, the culture medium is replaced with α-MEM complete medium containing 10% of the serum without exosomes, and the culture is performed for 48 hours. After the supernatant is collected and centrifuged, the precipitate is collected, resuspended with PBS, and exosomes RMC-Exos are obtained, which are stored at -80℃.

2. The method of claim 1, wherein the preparation of the Cervus elaphus Linnaeus stem cell exosome is characterized by, The gradient centrifugation method specifically comprises centrifugation at 1000 g for 5 minutes, centrifugation at 10000 g for 30 minutes, and ultracentrifugation at 100000 g for 4 hours.

3. The method of claim 1, wherein the preparation of the Cervus elaphus Linnaeus stem cell exosome is characterized by, The step of collecting the supernatant and performing centrifugation specifically comprises centrifugation at 1000 g for 5 minutes, centrifugation at 10000 g for 30 minutes, and ultracentrifugation at 100000 g for 4 hours.

4. A Cervus elaphus L stem cell exosome, characterized in that, It is prepared by the preparation method of any one of claims 1-3.

5. Use of the pilose antler stem cell exosomes of claim 4 in the preparation of a periodontal regeneration biomaterial.

6. Use according to claim 5, characterized in that, The pilose antler stem cell exosomes promote the proliferation, osteogenic differentiation, and alveolar bone regeneration of human gingival mesenchymal stem cells.

7. Use according to claim 5, characterized in that, The pilose antler stem cell exosomes are rich in vitronectin VTN.

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