Preparation method and application of a bone-inducing biological additive based on red blood cell-derived apoptotic vesicles

Abstract

The application discloses a preparation method of a bone formation inducing biological additive based on red blood cell-derived apoptotic vesicles and application thereof, and the preparation method comprises the following steps: a, red blood cell apoptosis induction treatment; b, collection and purification of red blood cell-derived apoptotic vesicles RBC-ApoEVs through series differential ultracentrifugation; c, characterization and quality control of the apoptotic vesicles; d, acquisition, culture and basic identification of SHED; and e, determination of 0.5 mu g / mL as the optimal bone formation inducing synergistic concentration of the red blood cell-derived apoptotic vesicles RBC-ApoEVs. The bone formation inducing biological additive prepared by the preparation method can be used as a safe and efficient bone formation inducing synergistic agent, and can solve the problem that "proliferation promotion" and "differentiation promotion" are difficult to be coordinated, and can also avoid the dependence on exogenous growth factors and related risks. The bone formation inducing biological additive can be applied to in-vitro induction of SHED bone differentiation, bone regeneration cell suspension and bone tissue engineering compound.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing an osteogenic induction bio-additive based on erythrocyte-derived apoptotic vesicles and its application. Background Technology

[0002] In the field of bone tissue regeneration, the use of stem cells for directed osteogenic differentiation is a core technical approach; among them, deciduous tooth pulp stem cells (SHED) are considered promising seed cells due to their strong proliferative and multi-directional differentiation potential.

[0003] Currently, the standard technique for inducing osteogenic differentiation of deciduous tooth pulp stem cells (SHEDs) in vitro involves using an osteogenic induction culture medium, whose core components typically include dexamethasone, sodium β-glycerophosphate, and ascorbic acid. While this standard technique is widely used, its efficacy and overall performance have clear limitations, primarily manifested in the following ways: 1. Bottleneck in induction efficiency: The osteogenic differentiation level that the formula of this standard technical solution can achieve has become stable. It is difficult to achieve a breakthrough in efficacy without changing the core components or significantly increasing the cost. This will result in the generated bone-like tissue failing to meet the complex repair needs in terms of speed, quantity or quality. 2. Potential inhibition of cell viability: During the induction of SHED differentiation of deciduous dental pulp stem cells, the proliferation rate of SHED deciduous dental pulp stem cells is often accompanied by a decrease, which leads to an insufficient total number of functional cells available for repair, affecting the final repair effect; 3. New problems introduced to improve efficacy: In pursuit of stronger effects, existing technologies often choose to add high doses of exogenous growth factors (such as BMP-2). Although this strategy can improve efficacy, it also significantly increases treatment costs and brings clinical risks such as heterotopic ossification, excessive bone resorption and inflammatory response. This makes it difficult to control the dosage and safety window of exogenous growth factors. 4. Lack of safe, stable and economical "synergists": Currently, there is a lack of bioactive additives that are seamlessly compatible with standard systems, have safe and stable sources, and are easy to prepare on a large scale, so as to synergistically enhance their dual effects of promoting proliferation and promoting bone formation without introducing new risks.

[0004] To address the aforementioned issues, there is an urgent need for an innovative biological additive that can be directly integrated with existing standard osteogenic induction protocols, significantly and safely improve overall performance (especially synergistically enhancing cell proliferation and osteogenic differentiation), and is industrially feasible. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing an osteogenic induction bio-additive based on erythrocyte-derived apoptotic vesicles and its application, addressing the shortcomings of existing technologies. The osteogenic induction bio-additive prepared by this method can synergistically enhance the proliferation activity, migration ability, and osteogenic differentiation efficiency of SHEDs without altering existing osteogenic induction culture media. This osteogenic induction bio-additive overcomes the limitation of existing technologies where it is difficult to simultaneously promote proliferation and differentiation, and effectively avoids dependence on exogenous growth factors and related risks. This osteogenic induction bio-additive achieves simultaneous enhancement of SHED's multidimensional functions through the addition of only a single component. This osteogenic induction bio-additive can be applied to in vitro induction of osteogenic differentiation of deciduous dental pulp stem cells (SHEDs), to bone regeneration cell suspensions, to the construction of collagen scaffold bone tissue engineering complexes, and to the construction of hydroxyapatite scaffold bone tissue engineering complexes.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions.

[0007] A method for preparing an osteogenic induction bio-additive based on erythrocyte-derived apoptotic vesicles includes the following steps: Step a, erythrocyte apoptosis induction treatment: Step a1: Collect fresh peripheral blood from a healthy donor using a vacuum blood collection tube containing K2EDTA anticoagulant; Step a2: Immediately place the fresh peripheral blood collected through the vacuum blood collection tube containing K2EDTA anticoagulant into a centrifuge for centrifugation, so that the fresh peripheral blood in the vacuum blood collection tube is separated into an upper layer containing plasma, white blood cells, and platelets and a lower layer containing red blood cells. Step a3: Collect the lower layer fluid containing red blood cells and separate the red blood cells; induced apoptosis of the separated red blood cells for 6 hours at 37°C using a blood mixer, and added 0.5 μM astrocytosine to the red blood cells; Step b: Collect and purify erythrocyte-derived apoptotic vesicles (RBCs-ApoEVs) using a series of differential ultracentrifugations. Step b1: The red blood cell suspension obtained after apoptosis-induced treatment with stellaria in step a3 is centrifuged in a centrifuge to obtain supernatant and precipitate. Then, the supernatant obtained after centrifugation is collected and transferred to an EP tube. Step b2: Centrifuge the supernatant in the EP tube to remove cells and debris from the supernatant. Step b3: Wash the precipitate obtained in step b1 twice with PBS. After each wash, centrifuge the precipitate to obtain purified red blood cell-derived apoptotic vesicles (RBC-ApoEVs). Step b4: Resuspend the purified red blood cell-derived apoptotic vesicles (RBC-ApoEVs) in 100 μL of PBS to obtain an RBC-ApoEVs resuspension, and store the RBC-ApoEVs resuspension at -80°C for later use. Step b5: Use the BCA protein assay kit to estimate the total protein concentration of the RBC-ApoEVs resuspension, and use this total protein concentration as the baseline for subsequent standardized addition. Step c: Characterization and quality control of apoptotic vesicles: Step c1: Observe the typical membrane structure of erythrocyte-derived apoptotic vesicles RBC-ApoEVs in the RBC-ApoEVs resuspension using cryo-transmission electron microscopy; determine the particle size distribution of erythrocyte-derived apoptotic vesicles RBC-ApoEVs by dynamic light scattering analysis. Step c2: Detect the expression of Caspase-3 protein in erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) by Western blotting; assess the expression of Annexin V in erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) using the Annexin V-FITC apoptosis detection kit; and determine the surface phosphatidylserine exposure level of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) by confocal microscopy and flow cytometry. Step d: Obtaining, culturing, and basic identification of deciduous tooth pulp stem cells (SHEDs): Step d1: Obtaining primary deciduous tooth pulp stem cells (SHED): Select healthy deciduous teeth extracted from children aged 6-8 years, and separate the pulp tissue from the selected deciduous teeth by enzymatic digestion to obtain primary deciduous tooth pulp stem cells (SHED). Step d2, SHED culture of primary deciduous tooth pulp stem cells: The obtained primary deciduous tooth pulp stem cells were cultured in vitro to passages P3-P5 for subsequent experiments; Step d3, Basic identification of deciduous tooth pulp stem cells SHED: Flow cytometry was used to verify the positive expression of mesenchymal stem cell surface markers CD90 and CD105 and the negative expression of hematopoietic markers CD34 and CD45 in deciduous tooth pulp stem cells SHED; osteogenic induction differentiation experiments, adipogenic induction differentiation experiments and chondrogenic induction differentiation experiments were used to verify the multi-lineage differentiation potential of deciduous tooth pulp stem cells SHED. Step e: Determine the optimal osteogenic induction synergistic concentration of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs): Different concentrations of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) were added to osteogenic induction medium to culture deciduous dental pulp stem cells (SHEDs) obtained in step b. Based on cell proliferation, cell migration, early osteogenic differentiation markers, expression of key osteogenic genes and proteins, and late osteogenic differentiation markers, 0.5 μg / mL was determined as the optimal osteogenic induction synergistic concentration of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs).

[0008] In step a2, the centrifuge speed is 1000×g, the centrifugation time is 10 minutes, and the centrifugation temperature is 4℃.

[0009] In step b1, the centrifuge speed is 1000×g, the centrifugation time is 10 minutes, and the centrifugation temperature is 4℃.

[0010] In step b2, the centrifuge speed is 16000×g, the centrifugation time is 30 minutes, and the centrifugation temperature is 4℃.

[0011] In step b3, the centrifuge speed is 16000×g, the centrifugation time is 30 minutes, and the centrifugation temperature is 4℃.

[0012] In step d2, the culture medium used for the in vitro culture of deciduous tooth pulp stem cells SHED is α-MEM medium containing 10% fetal bovine serum (FBS). The in vitro culture environment for SHED (shallow tooth pulp stem cells) was a constant temperature incubator at 37℃ and 5% CO2 concentration.

[0013] The osteogenic induction bio-additive prepared by the above-described method based on erythrocyte-derived apoptotic vesicles is applied to induce osteogenic differentiation of deciduous tooth pulp stem cells (SHED) in vitro. In the process of inducing osteogenic differentiation of deciduous dental pulp stem cells (SHED) in vitro, SHED were induced and cultured in an osteogenic induction medium containing erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

[0014] The osteogenic inducing biological additive prepared by the above-described method based on erythrocyte-derived apoptotic vesicles is applied to a bone regeneration cell suspension containing erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

[0015] The osteogenic induction bio-additive prepared by the above-described method based on erythrocyte-derived apoptotic vesicles is used to construct a collagen scaffold bone tissue engineering complex. During the construction of the collagen scaffold bone tissue engineering complex, a suspension of deciduous tooth pulp stem cells (SHED) was drop-inoculated onto the surface of the collagen scaffold. The SHED suspension contained erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

[0016] The osteogenic induction bio-additive prepared by the above-described method based on erythrocyte-derived apoptotic vesicles is used to construct a hydroxyapatite scaffold bone tissue engineering complex. During the construction of the hydroxyapatite scaffold bone tissue engineering complex, a suspension of deciduous tooth pulp stem cells (SHED) was drop-inoculated onto the surface of the hydroxyapatite scaffold. The SHED suspension contained erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

[0017] Compared with the prior art, the present invention has the following beneficial effects, specifically: 1. The osteogenic induction biological additive prepared by this invention can be directly compounded with the existing standard osteogenic induction culture medium without changing its core components, thereby safely and significantly improving the osteogenic induction efficacy of the standard system for SHED; at a concentration of 0.5 μg / mL, it can simultaneously enhance the proliferation activity and osteogenic differentiation index of SHED. 2. In the process of inducing osteogenic differentiation of deciduous tooth pulp stem cells (SHED) in vitro, by adding the osteogenic induction biological additive prepared or prepared in this invention, the standard induction system can simultaneously enhance the proliferation and migration ability of stem cells while driving SHED differentiation, thereby obtaining a more abundant number of osteogenic cells with more mature functions at the differentiation endpoint, overcoming the problem of the difficulty in synergistic "proliferation promotion" and "differentiation promotion" in the existing technology. 3. This invention can reduce or avoid the need to add high doses, high costs, and high risks of exogenous growth factors to improve induction efficiency, thereby reducing treatment costs and improving the safety of clinical applications; 4. The osteogenic induction biological additives prepared or manufactured in this invention are suitable for preparing bone regeneration cell suspensions or for constructing bone tissue engineering complexes with collagen scaffolds and hydroxyapatite scaffolds. Attached Figure Description

[0018] The present invention will be further described below with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the present invention.

[0019] Figure 1This is a schematic diagram of the procedure for isolating RBC-ApoEVs from human erythrocytes.

[0020] Figure 2 Characterization of RBC-ApoEVs.

[0021] Figure 3 This refers to the endocytosis of RBC-ApoEVs by SHED.

[0022] Figure 4 RBC-ApoEVs promote SHED proliferation.

[0023] Figure 5 To facilitate SHED migration in RBC-ApoEVs.

[0024] Figure 6 RBC-ApoEVs promote SHED osteoogenesis.

[0025] Figure 7 Transcriptome analysis of SHED treated with 0.5 μg / mL RBC-ApoEVs and untreated SHEDs.

[0026] Figure 8 Osteogenic gene expression profiles in SHEDs treated with 0.5 μg / mL RBC-ApoEVs for 7 days. Detailed Implementation

[0027] The present invention will now be described in conjunction with specific embodiments.

[0028] Example 1: A method for preparing an osteogenic induction bio-additive based on erythrocyte-derived apoptotic vesicles, comprising the following steps: Step a, erythrocyte apoptosis induction treatment: Step a1, as follows Figure 1 As shown in Figure A, fresh peripheral blood was collected from a healthy donor using a vacuum blood collection tube containing K2EDTA anticoagulant. The inclusion criteria for donors were: 1. Age 20-30 years; 2. Non-smoker; 3. Good health and no infectious diseases; 4. No blood diseases and normal platelet count; 5. No medications affecting platelet function within the three months prior to donation. All participants received written informed consent before blood collection. Step a2, as follows Figure 1 As shown in B, fresh peripheral blood collected through a vacuum blood collection tube containing K2EDTA anticoagulant is immediately placed in a centrifuge for centrifugation, so that the fresh peripheral blood in the vacuum blood collection tube is separated into an upper layer containing plasma, white blood cells, and platelets, and a lower layer containing red blood cells. Step a3, as follows Figure 1As shown in Figure C, the lower layer fluid containing red blood cells was collected and the red blood cells were separated. The separated red blood cells were subjected to apoptosis-induced treatment for 6 hours at 37°C using a blood mixer (Zuole, Shanghai, China), and 0.5 μM staphylococcal (STS; Cell Signaling Technology, USA) was added to the red blood cells. Step b: Collect and purify erythrocyte-derived apoptotic vesicles (RBCs-ApoEVs) using a series of differential ultracentrifugations. Step b1, as follows Figure 1 As shown in D, the red blood cell suspension obtained after apoptosis-induced treatment with stellaria in step a3 is centrifuged in a centrifuge to obtain supernatant and precipitate. Then, the supernatant obtained after centrifugation is collected and transferred to an EP tube. Step b2: Centrifuge the supernatant in the EP tube to remove cells and debris from the supernatant. Step b3, as follows Figure 1 E and Figure 1 As shown in F, the precipitate obtained in step b1 was washed twice with PBS, and after each wash, it was centrifuged to obtain purified erythrocyte-derived apoptotic vesicles (RBC-ApoEVs). Step b4: Resuspend the purified red blood cell-derived apoptotic vesicles (RBC-ApoEVs) in 100 μL of PBS to obtain an RBC-ApoEVs resuspension, and store the RBC-ApoEVs resuspension at -80°C for later use. Step b5: Use the BCA protein assay kit (Beijing Tiangen, China) to estimate the total protein concentration of the RBC-ApoEVs resuspension and use this total protein concentration as the baseline for subsequent standardized addition. Step c: Characterization and quality control of apoptotic vesicles: Step c1: Observe the typical membranous structure of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) in the RBC-ApoEVs resuspension using cryo-transmission electron microscopy, such as... Figure 2 As shown in Figure A, Cryo-TEM images reveal that RBC-ApoEVs are circular in shape and vary in size: larger apoptotic vesicles (approximately 400 nm, black arrows) and smaller apoptotic vesicles (approximately 80 nm, white arrows). The particle size distribution of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) was determined by dynamic light scattering analysis, as shown in Figure A. Figure 2 As shown in B, the particle size distribution obtained by DLS analysis shows two distinct peaks: a minor peak (approximately 80 nm, intensity 6%) and a major peak (approximately 400 nm, intensity 27%). Step c2: Detect the expression of Caspase-3 protein in erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) using Western blotting (e.g., ...). Figure 2 As shown in Figure C: Western blot analysis of caspase-3 expression in RBC-ApoEVs); Annexin V expression in erythrocyte-derived apoptotic vesicles RBC-ApoEVs was assessed using the Annexin V-FITC apoptosis detection kit (7Sea Biotech, Shanghai, China). Figure 2 As shown in D, phosphatidylserine exposure was demonstrated by Annexin V staining (green); the surface phosphatidylserine exposure level of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) was determined by confocal microscopy and flow cytometry. Step c3: PKH26-labeled erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) (based on protein content, e.g., 200 μg / mL) were co-cultured with deciduous tooth pulp stem cells (SHEDs) for 6 h, 12 h, and 24 h. Confocal microscopy was used to observe and confirm that the vesicles were effectively internalized by SHEDs within 24 hours. The results are as follows: Figure 3 As shown; Step d: Obtaining, culturing, and basic identification of deciduous tooth pulp stem cells (SHEDs): Step d1: Obtaining primary deciduous tooth pulp stem cells (SHED): Select healthy deciduous teeth extracted from children aged 6-8 years, and separate the pulp tissue from the selected deciduous teeth by enzymatic digestion to obtain primary deciduous tooth pulp stem cells (SHED). Step d2, SHED culture of primary deciduous tooth pulp stem cells: The obtained primary deciduous tooth pulp stem cells were cultured in vitro to passages P3-P5 for subsequent experiments; Step d3, Basic identification of deciduous tooth pulp stem cells SHED: Flow cytometry was used to verify the positive expression of mesenchymal stem cell surface markers CD90 and CD105 and the negative expression of hematopoietic markers CD34 and CD45 in deciduous tooth pulp stem cells SHED; osteogenic induction differentiation experiments, adipogenic induction differentiation experiments and chondrogenic induction differentiation experiments were used to verify the multi-lineage differentiation potential of deciduous tooth pulp stem cells SHED. Step e: Determine the optimal osteogenic induction synergistic concentration of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs): Different concentrations of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) were added to osteogenic induction medium to culture deciduous dental pulp stem cells (SHEDs) obtained in step b. Based on cell proliferation, cell migration, early osteogenic differentiation markers, expression of key osteogenic genes and proteins, and late osteogenic differentiation markers, 0.5 μg / mL was determined as the optimal osteogenic induction synergistic concentration of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs).

[0029] like Figure 4 As shown, RBC-ApoEVs (0.5 μg / mL, 1 μg / mL, 2 μg / mL) stimulated SHED proliferation on days 5 and 7. The results were assessed by the CCK-8 assay (n=6). The difference was significant compared with 0 μg / mL RBC-ApoEVs (*p<0.01).

[0030] like Figure 5 As shown in Figure A, representative Transwell chamber experimental images demonstrate the migration of SHEDs after treatment with RBC-ApoEVs (0.5 μg / mL, 1 μg / mL, 2 μg / mL) for 24 h; Figure 5 As shown in Figure B, quantitative analysis of the average number of migrating cells per field of view at 24 h time points; as shown in Figure B. Figure 5 As shown in Figure C, the scratch test images show the wound healing status 24 h after treatment with RBC-ApoEVs (0.5 μg / mL, 1 μg / mL, 2 μg / mL); Figure 5 As shown in Figure D, ImageJ software was used to quantitatively analyze wound area recovery (n=3). The difference was significant compared to 0 μg / mL RBC-ApoEVs (*p<0.01).

[0031] In step a2, the centrifuge speed is 1000×g, the centrifugation time is 10 minutes, and the centrifugation temperature is 4℃.

[0032] In addition, in step b1, the centrifuge speed is 1000×g, the centrifugation time is 10 minutes, and the centrifugation temperature is 4℃.

[0033] Furthermore, in step b2, the centrifuge speed is 16000×g, the centrifugation time is 30 minutes, and the centrifugation temperature is 4℃.

[0034] It should be noted that in step b3, the centrifuge speed is 16000×g, the centrifugation time is 30 minutes, and the centrifugation temperature is 4℃.

[0035] It should be further noted that, in step d2, the culture medium used for the in vitro culture of deciduous tooth pulp stem cells SHED is α-MEM medium containing 10% fetal bovine serum (FBS). The in vitro culture environment for SHED (shallow tooth pulp stem cells) was a constant temperature incubator at 37℃ and 5% CO2 concentration.

[0036] Specifically, in step e, when determining the optimal osteogenic induction synergistic concentration of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) based on cell proliferation, cell migration, early osteogenic differentiation markers, expression of key osteogenic genes and proteins, and late osteogenic differentiation markers, the specific procedure is as follows: 1. Cell proliferation: Cell viability of deciduous dental pulp stem cells cultured with different concentrations of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) on days 1, 3, 5, and 7 was determined using the CCK-8 assay to identify the optimal concentration range for promoting proliferation. 2. Cell migration: The migration ability of SHED stem cells from deciduous teeth pulp at different concentrations was evaluated by scratch assay and Transwell assay. 3. Early markers of osteogenic differentiation: Deciduous tooth pulp stem cells (DPS) cultured with different concentrations of erythrocyte-derived apoptotic vesicle (RBC-ApoEVs) on days 4 and 7 were analyzed using alkaline phosphatase (ALP) activity detection and staining to determine the concentration of ALP with the strongest expression. Results are as follows: Figure 6 A and Figure 6 As shown in B; 4. Key Osteogenic Genes and Protein Expression: The expression levels of genes such as RUNX2, COL-1, OPN, and OCN, and proteins such as ALP, RUNX2, and COL-1, were detected by RT-qPCR and Western Blot on days 7 and 14 of SHED culture of deciduous dental pulp stem cells from different concentrations of erythrocyte-derived apoptotic vesicle (RBC-ApoEVs). The results are as follows: Figure 6 C and Figure 6 As shown in D; 5. Late markers of osteogenic differentiation: Deciduous tooth pulp stem cells cultured for 16 days with different concentrations of erythrocyte-derived apoptotic vesicle (RBC-ApoEVs) via SHED were analyzed by Alizarin Red S staining and quantitative determination of calcium nodule formation. The results are as follows: Figure 6 As shown in E and 6F.

[0037] It is important to emphasize that SHED (Self-Derived Dental Pulp Stem Cells) derived from erythrocytes at the optimal osteogenic induction synergistic concentration (0.5 μg / mL) and treated with standard osteogenic induction medium for a certain period (day 7) were used for transcriptome sequencing (RNA-seq). Differentially expressed genes were screened using bioinformatics analysis, and pathway enrichment analysis was performed using databases such as KEGG to reveal key synergistic signaling pathways (such as HIF-1 and PI3K-Akt pathways). Core genes in these pathways (such as VEGFA and IL7R) were validated using RT-qPCR. Specifically, for example... Figure 7Figure A shows a heatmap of DEGs between the control group (0 μg / mL RBC-ApoEVs) and the treatment group (0.5 μg / mL RBC-ApoEVs) (|log2FC| ≥1, q <0.05). Columns: biological replicates (n=3 per group); rows: genes; color scale: log10(FPKM+1e-6) (red: upregulated; blue: downregulated). Figure 7 As shown in B, the PCA plot reveals clear clustering of the transcriptome profile (95% confidence ellipse). Figure 7 As shown in C, the volcano plot of DEGs (red: upregulation; blue: downregulation; |log2FC| ≥1, corrected p < 0.05); dashed line: significance threshold (|log2FC|=1, corrected p=0.05). Figure 7 As shown in Figure D, GO enrichment analysis of DEGs in three ontology categories: cellular composition, molecular function, and biological processes. Figure 7 E shows the top 20 enriched KEGG pathways sorted by p-value. Additionally, as... Figure 8 As shown in Figure A, a heatmap of the top 20 osteogenic DEGs between the control group (0 μg / mL RBC-ApoEVs) and the treatment group (0.5 μg / mL RBC-ApoEVs) (|log2FC| ≥ 2.5, q < 0.05). Figure 8 As shown in B, the top 10 enriched osteogenic-related KEGG pathways; Y-axis: KEGG pathways; X-axis: number of DEGs (top) and enrichment significance (bottom). Figure 8 As shown in Figure C, the GSEA diagram of the HIF-1 and PI3K-Akt signal paths; as follows. Figure 8 As shown in D, seven upregulated genes selected from the top 20 osteogenic DEGs are displayed; as... Figure 8 E shows the RT-qPCR analysis of osteogenic-related gene expression; there were significant differences compared with 0 μg / mLRBC-ApoEVs, *p<0.05 and **p<0.01.

[0038] In summary, the preparation method of the osteogenic induction biological additive based on erythrocyte-derived apoptotic vesicles in Example 1 has the following technical advantages, specifically: 1. The osteogenic induction biological additive prepared in Example 1 can be used as a safe and efficient osteogenic induction synergist. It can be directly combined with the existing standard osteogenic induction culture medium without changing its core components, thereby safely and significantly improving the osteogenic induction efficacy of the standard system for deciduous tooth pulp stem cells (SHED). Without changing the core components of the standard osteogenic induction culture medium, only a trace amount (0.5 μg / mL) of RBC-ApoEVs needs to be added to achieve synergistic effects. Experimental data show that this compound system increased the amount of mineralized nodules formed—the osteogenic differentiation endpoint of SHED—by 2.5 times, while the expression levels of key osteogenic genes (such as OCN and RUNX2) and proteins were significantly and synchronously upregulated. 2. The osteogenic induction biological additive prepared in Example 1 effectively solves the problem of the difficulty in synergistically promoting proliferation and differentiation. During the in vitro induction of osteogenic differentiation of deciduous dental pulp stem cells (SHEDs), the addition of the osteogenic induction biological additive prepared in Example 1 enables the standard induction system to simultaneously enhance the proliferation and migration capabilities of SHEDs while driving their differentiation. This results in a more abundant number and more functionally mature osteogenic cell at the differentiation endpoint, overcoming the limitation of existing technologies where both aspects are difficult to achieve simultaneously. In the induction system containing RBC-ApoEVs, the proliferation capacity (days 5-7) and migration activity (up to 1.8-fold increase within 24 hours) of SHEDs are significantly enhanced, while their osteogenic differentiation program is strongly driven. This solves the problem of cell proliferation inhibition often associated with induced differentiation in existing technologies, ensuring a more abundant number and more functionally mature osteogenic cell at the differentiation endpoint, providing a higher quality cell source for subsequent tissue repair. 3. The osteogenic induction biological additive prepared in Example 1 can effectively avoid dependence on exogenous growth factors and related risks. The osteogenic induction biological additive prepared in Example 1 can provide an alternative synergistic solution that is derived from the human body, has a clear composition, and is safe and controllable. This reduces or avoids the need to add high doses, high costs, and high risks of exogenous growth factors (such as BMP-2) to improve induction efficiency, thereby reducing treatment costs and improving the safety of clinical application. 4. The preparation method of osteogenic induction biological additive based on erythrocyte-derived apoptotic vesicles in Example 1 provides a standardized preparation, characterization and application method for erythrocyte-derived apoptotic vesicles (RBC-ApoEVs). This enables RBC-ApoEVs to be seamlessly integrated into existing stem cell culture and tissue engineering product production processes as a stable, scalable, and ready-to-use culture medium additive, promoting the clinical translation of bone regeneration technology.

[0039] Example 2: An osteogenic induction bio-additive prepared by the above-described method for preparing osteogenic induction bio-additive based on apoptotic vesicles derived from erythrocytes is applied to induce osteogenic differentiation of deciduous tooth pulp stem cells (SHED) in vitro. In the process of inducing osteogenic differentiation of deciduous dental pulp stem cells (SHED) in vitro, SHED were induced and cultured in an osteogenic induction medium containing erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

[0040] Example 3: An osteogenic inducing biological additive prepared by the method described above based on erythrocyte-derived apoptotic vesicles is applied to a bone regeneration cell suspension containing erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

[0041] Example 4: An osteogenic inducing bio-additive prepared by the above-described method based on erythrocyte-derived apoptotic vesicles is applied to the construction of a collagen scaffold bone tissue engineering complex. During the construction of the collagen scaffold bone tissue engineering complex, a suspension of deciduous tooth pulp stem cells (SHED) was drop-inoculated onto the surface of the collagen scaffold. The SHED suspension contained erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

[0042] Example 5: An osteogenic inducing bio-additive prepared by the above-described method based on erythrocyte-derived apoptotic vesicles is applied to the construction of a hydroxyapatite scaffold bone tissue engineering complex. During the construction of the hydroxyapatite scaffold bone tissue engineering complex, a suspension of deciduous tooth pulp stem cells (SHED) was drop-inoculated onto the surface of the hydroxyapatite scaffold. The SHED suspension contained erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

[0043] The above description is only a preferred embodiment of the present invention. For those skilled in the art, there will be changes in the specific implementation and application scope based on the ideas of the present invention. The content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing an osteogenic inducing biological additive based on erythrocyte-derived apoptotic vesicles, characterized in that, It includes the following steps, specifically: Step a, erythrocyte apoptosis induction treatment: Step a1: Collect fresh peripheral blood from a healthy donor using a vacuum blood collection tube containing K2EDTA anticoagulant; Step a2: Immediately place the fresh peripheral blood collected through the vacuum blood collection tube containing K2EDTA anticoagulant into a centrifuge for centrifugation, so that the fresh peripheral blood in the vacuum blood collection tube is separated into an upper layer containing plasma, white blood cells, and platelets and a lower layer containing red blood cells. Step a3: Collect the lower layer fluid containing red blood cells and separate the red blood cells; induced apoptosis of the separated red blood cells for 6 hours at 37°C using a blood mixer, and added 0.5 μM astrocytosine to the red blood cells; Step b: Collect and purify erythrocyte-derived apoptotic vesicles (RBCs-ApoEVs) using a series of differential ultracentrifugations. Step b1: The red blood cell suspension obtained after apoptosis-induced treatment with stellaria in step a3 is centrifuged in a centrifuge to obtain supernatant and precipitate. Then, the supernatant obtained after centrifugation is collected and transferred to an EP tube. Step b2: Centrifuge the supernatant in the EP tube to remove cells and debris from the supernatant. Step b3: Wash the precipitate obtained in step b1 twice with PBS. After each wash, centrifuge the precipitate to obtain purified red blood cell-derived apoptotic vesicles (RBC-ApoEVs). Step b4: Resuspend the purified red blood cell-derived apoptotic vesicles (RBC-ApoEVs) in 100 μL of PBS to obtain an RBC-ApoEVs resuspension, and store the RBC-ApoEVs resuspension at -80°C for later use. Step b5: Use the BCA protein assay kit to estimate the total protein concentration of the RBC-ApoEVs resuspension, and use this total protein concentration as the baseline for subsequent standardized addition. Step c: Characterization and quality control of apoptotic vesicles: Step c1: Observe the typical membrane structure of erythrocyte-derived apoptotic vesicles RBC-ApoEVs in the RBC-ApoEVs resuspension using cryo-transmission electron microscopy; determine the particle size distribution of erythrocyte-derived apoptotic vesicles RBC-ApoEVs by dynamic light scattering analysis. Step c2: Detect the expression of Caspase-3 protein in erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) by Western blotting; assess the expression of Annexin V in erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) using the Annexin V-FITC apoptosis detection kit; and determine the surface phosphatidylserine exposure level of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) by confocal microscopy and flow cytometry. Step d: Obtaining, culturing, and basic identification of deciduous tooth pulp stem cells (SHEDs): Step d1: Obtaining primary deciduous tooth pulp stem cells (SHED): Select healthy deciduous teeth extracted from children aged 6-8 years, and separate the pulp tissue from the selected deciduous teeth by enzymatic digestion to obtain primary deciduous tooth pulp stem cells (SHED). Step d2, SHED culture of primary deciduous tooth pulp stem cells: The obtained primary deciduous tooth pulp stem cells were cultured in vitro to passages P3-P5 for subsequent experiments; Step d3, Basic identification of deciduous tooth pulp stem cells SHED: Flow cytometry was used to verify the positive expression of mesenchymal stem cell surface markers CD90 and CD105 and the negative expression of hematopoietic markers CD34 and CD45 in deciduous tooth pulp stem cells SHED; osteogenic induction differentiation experiments, adipogenic induction differentiation experiments and chondrogenic induction differentiation experiments were used to verify the multi-lineage differentiation potential of deciduous tooth pulp stem cells SHED. Step e: Determine the optimal osteogenic induction synergistic concentration of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs): Different concentrations of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) were added to osteogenic induction medium to culture deciduous dental pulp stem cells (SHEDs) obtained in step b. Based on cell proliferation, cell migration, early osteogenic differentiation markers, expression of key osteogenic genes and proteins, and late osteogenic differentiation markers, 0.5 μg / mL was determined as the optimal osteogenic induction synergistic concentration of erythrocyte-derived apoptotic vesicles (RBC-ApoEVs).

2. The method for preparing an osteogenic induction bio-additive based on erythrocyte-derived apoptotic vesicles according to claim 1, characterized in that: In step a2, the centrifuge speed is 1000×g, the centrifugation time is 10 minutes, and the centrifugation temperature is 4℃.

3. The method for preparing an osteogenic induction bio-additive based on erythrocyte-derived apoptotic vesicles according to claim 1, characterized in that: In step b1, the centrifuge speed is 1000×g, the centrifugation time is 10 minutes, and the centrifugation temperature is 4℃.

4. The method for preparing an osteogenic induction bio-additive based on erythrocyte-derived apoptotic vesicles according to claim 1, characterized in that: In step b2, the centrifuge speed is 16000×g, the centrifugation time is 30 minutes, and the centrifugation temperature is 4℃.

5. The method for preparing an osteogenic induction bio-additive based on erythrocyte-derived apoptotic vesicles according to claim 1, characterized in that: In step b3, the centrifuge speed is 16000×g, the centrifugation time is 30 minutes, and the centrifugation temperature is 4℃.

6. The method for preparing an osteogenic induction bio-additive based on erythrocyte-derived apoptotic vesicles according to claim 1, characterized in that: In step d2, the culture medium used for the in vitro culture of deciduous tooth pulp stem cells (SHED) is α-MEM medium containing 10% fetal bovine serum (FBS). The in vitro culture environment for SHED (shallow tooth pulp stem cells) was a constant temperature incubator at 37℃ and 5% CO2 concentration.

7. The osteogenic inducing biological additive prepared by the method of any one of claims 1 to 6 based on erythrocyte-derived apoptotic vesicles is applied to induce osteogenic differentiation of deciduous tooth pulp stem cells (SHED) in vitro. In the process of inducing osteogenic differentiation of deciduous dental pulp stem cells (SHED) in vitro, SHED were induced and cultured in an osteogenic induction medium containing erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

8. The osteogenic inducing biological additive prepared by the method of any one of claims 1 to 6 based on erythrocyte-derived apoptotic vesicles is applied to a bone regeneration cell suspension containing erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

9. The osteogenic inducing bio-additive prepared by the method of any one of claims 1 to 6 based on erythrocyte-derived apoptotic vesicles is used to construct a collagen scaffold bone tissue engineering complex; During the construction of the collagen scaffold bone tissue engineering complex, a suspension of deciduous tooth pulp stem cells (SHED) was drop-inoculated onto the surface of the collagen scaffold. The SHED suspension contained erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

10. The osteogenic inducing bio-additive prepared by the method of any one of claims 1 to 6 based on erythrocyte-derived apoptotic vesicles is used to construct a hydroxyapatite scaffold bone tissue engineering complex. During the construction of the hydroxyapatite scaffold bone tissue engineering complex, a suspension of deciduous tooth pulp stem cells (SHED) was drop-inoculated onto the surface of the hydroxyapatite scaffold. The SHED suspension contained erythrocyte-derived apoptotic vesicles (RBC-ApoEVs) at a concentration of 0.5 μg / mL.

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