Bioactive material for inducing angiogenesis and osteogenesis as well as preparation method and application of bioactive material

Abstract

The invention discloses a bioactive material for inducing blood vessel and osteogenesis and a preparation method and application of the bioactive material, and belongs to the technical field of tissue engineering, the bioactive material for inducing blood vessel and osteogenesis comprises Schneid membrane mesenchymal stem cells and platelet-rich fibrous protein membranes, the Schneider membrane mesenchymal stem cells are loaded on the surface of the platelet-rich fibrin membrane, and the Schneider membrane mesenchymal stem cells have the strong membrane forming characteristic when cultured in vitro, can gather and proliferate massively on the surface of PRF, continuously secrete an extracellular matrix to wrap the PRF and replace an original stent along with degradation of the PRF, so that the Schneider membrane mesenchymal stem cells can be used for preparing the stent. The SMMSCs can continuously release bioactive factors, so that the osteogenic function period of the material is prolonged. In addition, the PRF membrane loaded with SMMSCs widens the application range of PRF, can be directly used as a bone grafting filling material, and also can be used as a periosteum-like guided tissue regeneration barrier membrane.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, in particular to a bioactive material inducing angiogenesis and bone formation and a preparation method and application thereof. Background Art

[0002] The repair of periodontal bone defects has always been a difficult problem in clinical treatment. In severe cases, it leads to tooth loss and difficulty in dentition repair. At present, the main clinical treatment methods include autologous bone transplantation and artificial bone replacement materials. Autologous bone is taken from the patient himself, causing the trauma of secondary surgery, and the risk of postoperative infection is high. Artificial bone transplant materials are often used as the main clinical application materials to replace autologous bone, such as deproteinized bovine bone mineral (DBBM), etc. Their osteogenesis effect is relatively stable, but they may also cause problems such as rejection and infection, and their osteoinductivity is relatively poor compared to autologous bone, resulting in a long bone regeneration cycle. The repair of periodontal bone defects has certain particularities. The shape of the bone defect site is irregular, and the periodontal bone defect area is not easy to be fully filled with hard osteogenic materials. In order to protect the adjacent healthy periodontal tissue from trauma as much as possible, minimally invasive surgery is required. Therefore, the existing hard bone transplant materials lack specificity and accuracy.

[0003] Tissue engineering technology is an important strategy to achieve bone regeneration. The construction of tissue-engineered bone mainly includes scaffold materials, seed cells and cytokines. Platelet-rich fibrin (PRF) is an autologous transplant material composed of a dense fibrin network and rich in a variety of growth factors and cytokines. It is a kind of autologous platelet concentrate (APC) and a soft transplant material that can match the deformation of the defect area. PRF has been widely used in clinical practice, mainly for promoting the repair and regeneration of defects in various tissues and organs. PRF contains a variety of growth factors that promote cell proliferation and migration, including PDGF-BB, VEGF, FGF, EGF and TGF-β, which can effectively promote angiogenesis, wound healing and connective tissue increment. However, the role of PRF in bone tissue regeneration is still controversial, mainly because the degradation rate of fibrin is too fast and does not match the osteogenic cycle, which limits it to releasing growth factors and cytokines in the early osteogenic microenvironment and plays a limited role in bone induction regulation. Therefore, it is necessary to functionally modify PRF and prolong its effect time in osteogenesis.

[0004] Mesenchymal stem cells (MSCs) are seed cells for tissue engineering technology. They have low immunogenicity, multidirectional differentiation and immune regulation. They are an important cell therapy strategy in the field of regenerative medicine and have high practicality in clinical transformation. MSCs from different tissue types show different transcriptome patterns and differentiation potentials, which make them play different roles in tissue healing and regeneration. The epigenetic state of MSCs is related to the lineage plasticity of their tissue of origin, involving mechanisms related to the maintenance of epigenetic memory. Schneiderian's membrane derived mesenchymal stem cells (SMMSCs) are a type of MSCs that have been discovered and continuously studied in the field of oral maxillary sinus bone regeneration in recent years. Due to the particularity of their source, the cells proliferate rapidly when cultured in vitro, are easy to grow in layers and form a membrane structure, and do not undergo contact inhibition of apoptosis between cells. Therefore, they are more suitable as seed cells for tissue engineering scaffolds.

[0005] The loading methods of seed cells on tissue engineering scaffolds are mainly divided into two forms: encapsulation inside the scaffold material and external loading on the surface of the scaffold material. Studies have shown that the external loading method is more adapted to the biological behavior of cells, which is conducive to the proliferation, migration and osteogenic differentiation of MSCs, secretion of growth factors and cytokines and effective release to the surrounding to recruit host cells to participate in the construction of the osteogenic microenvironment. The three-dimensional loose reticular structure of PRF can provide a suitable three-dimensional growth space for the adhesion and proliferation of MSCs on its surface. And studies have shown that the components of PRF extract can effectively promote the osteogenic differentiation of SMMSCs. Therefore, PRF can be used as an in vivo transplantation carrier of SMMSCs and as a transformation application form of SMMSCs for the treatment of bone defects. At the same time, MSCs loaded on the surface of PRF can be activated by the growth factors released by PRF, continue to proliferate and tend to osteogenic differentiation, secrete osteogenic-related small molecule proteins and bone matrix proteins, and continue to play a biological function as the fibrin in PRF degrades and replaces the position of the material itself, which is equivalent to prolonging the in vivo retention time of the transplant and enhancing its osteogenic performance. In summary, there are many shortcomings in the use of PRF alone for bone defect repair. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] Therefore, the object of the present invention is to provide a bioactive material for inducing angiogenesis and bone formation, and a preparation method and application thereof, so as to solve the problems raised in the above background technology.

[0008] To solve the above technical problems, according to one aspect of the present invention, the present invention provides the following technical solutions: A bioactive material for inducing angiogenesis and osteogenesis comprises: Schneider membrane mesenchymal stem cells and platelet-rich fibrin membrane sheets, wherein the Schneider membrane mesenchymal stem cells are loaded on the surface of the platelet-rich fibrin membrane sheets.

[0009] As a preferred embodiment of the bioactive material for inducing angiogenesis and osteogenesis of the present invention, the Schneider membrane mesenchymal stem cells include mesenchymal stem cells derived from the mucoperiosteum layer of the maxillary sinus and mesenchymal stem cells derived from the lamina propria.

[0010] A method for preparing a bioactive material that induces angiogenesis and osteogenesis, comprising the following steps: S1. Place a platelet-rich fibrin membrane obtained from 10 ml of venous blood in a 24-well plate and add 1×10 5 Schneider membrane mesenchymal stem cells were seeded on the surface of a platelet-rich fibrin membrane, and the complete culture medium was α-MEM medium containing 10% fetal bovine serum + 1% penicillin-streptomycin; S2. After 24 h of co-culture, replace the medium with osteogenic induction medium, which is a complete medium containing 0.01 M β-sodium glycerophosphate + 0.25 mM ascorbic acid + 100 nM dexamethasone. Continue co-culture until the 7th day for use. During this period, the osteogenic induction medium is fully replaced every 2 days.

[0011] As a preferred embodiment of the method for preparing a bioactive material that induces angiogenesis and osteogenesis according to the present invention, in step S1, Schneider membrane mesenchymal stem cells are obtained by the following steps: The obtained 1×1 cm healthy human maxillary sinus mucosal tissue was immersed in sterile phosphate buffer containing 10% penicillin-streptomycin, temporarily stored on ice, and subsequently processed within 2 h; The periosteum layer of the maxillary sinus mucosa was torn off and separated into tissue blocks no larger than 0.3×0.3 cm and placed in a T25 culture flask for in vitro culture of PMSCs. The remaining maxillary sinus mucosa tissue was placed in 2 ml of dispase neutral protease at 37°C to digest and remove the epithelial layer. The remaining periosteum layer was washed with PBS and separated into tissue blocks no larger than 0.3×0.3 cm and placed in a T25 culture flask for in vitro culture of LMSCs. The complete culture medium consisted of α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The culture conditions were 37°C and 5% CO 2 , replace the medium every 2-3 days until the cell confluence reaches 80%-90% for subculturing; The cells were digested with 0.25% trypsin solution and terminated with twice the volume of culture medium containing 10% fetal bovine serum. The cells were collected by centrifugation at 300 g for 5 min and inoculated into new culture flasks according to the cell confluence of 50%-60%; Then cryopreservation is carried out. During cryopreservation, the cells are digested and centrifuged, the supernatant is removed, and cell freezing solution is added, with a ratio of 92% dimethyl sulfoxide + 8% fetal bovine serum. The cells are preserved using the gradient freezing method. When reviving, the cells are taken out and quickly placed in a 37°C water bath until the liquid melts. After adding 9 times the volume of complete culture medium and mixing evenly, the cells are collected by centrifugation at 300g for 5 minutes and inoculated into new culture bottles according to the cell confluence of 60%-70%.

[0012] As a preferred embodiment of the method for preparing a bioactive material that induces angiogenesis and osteogenesis according to the present invention, in step S1, the platelet-rich fibrin membrane is obtained by the following steps: 10 ml of blood was drawn from the elbow vein and placed in a sterile thrombin-free blood collection tube, which was immediately centrifuged at 3000 rpm for 10 min. After standing, three layers appeared in the tube, from the bottom to the top, namely the red blood cell layer, the white gel-like PRF layer, and the platelet-poor serum layer. Use sterile tweezers to pinch the middle PRF layer and place it on the surface of three layers of sterile gauze. Cover the three layers of gauze and press gently and slowly to remove moisture from the PRF gel block to obtain a platelet-rich fibrin membrane sheet.

[0013] A bioactive material that induces angiogenesis and osteogenesis and serves as a barrier membrane for guiding tissue regeneration in the field of bone regeneration.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention utilizes the strong film-forming properties of Schneider membrane mesenchymal stem cells (SMMSCs) when cultured in vitro. They can aggregate and proliferate in large quantities on the surface of PRF, continuously secrete extracellular matrix to wrap PRF, and replace the original scaffold as PRF degrades. SMMSCs can continuously release bioactive factors, thereby extending the bone formation function cycle of the material. In addition, the PRF membrane loaded with SMMSCs broadens the application range of PRF. In addition to being directly used as a bone graft filling material, it can also be used as a barrier membrane similar to periosteum to guide tissue regeneration.

[0015] 2. The present invention solves the problem of administration routes for stem cell therapy in the field of bone regeneration. Specifically, the PRF membrane can serve as a natural niche for mesenchymal stem cells (MSCs) to enhance their biological activity and osteogenic function. When cells are loaded on the PRF surface, the cells are better stretched and more effective cytokine release is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below in combination with the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them: Figure 1 The morphological images of two types of mesenchymal stem cells LMSCs and PMSCs derived from Schneider membrane provided by the present invention under an optical microscope; Figure 2 The staining diagram for identification of osteogenic, adipogenic and chondrogenic three-way differentiation of two types of mesenchymal stem cells LMSCs and PMSCs derived from Schneider membrane provided by the present invention; Figure 3 Flow cytometric identification of stem cell surface markers of two types of mesenchymal stem cells LMSCs and PMSCs derived from Schneider membrane provided by the present invention; Figure 4 The PRF gel and membrane provided by the present invention; Figure 5 H&E staining of histological sections of PRF loaded with LMSCs and PMSCs provided by the present invention; Figure 6 The tissue immunofluorescence staining diagram of the PRF loaded with LMSCs and PMSCs provided by the present invention expressing the angiogenesis-related protein VEGF and the osteogenic-related protein OCN, wherein DAPI is used for cell nucleus staining; Figure 7 The tissue immunofluorescence staining diagram of the expression of angiogenesis-related protein VEGF and osteogenic-related protein OCN by PRF loaded with LMSCs and PMSCs provided by the present invention after ectopic transplantation in vivo, wherein Dil is used to mark the cell membrane of LMSCs and PMSCs; Figure 8 Single cell sequencing cell typing and ratio diagram of PRF loaded with LMSCs and PMSCs provided by the present invention; Fig. 9 A schematic flow chart of the method for producing bioactive materials that induce angiogenesis and osteogenesis provided by the present invention. DETAILED DESCRIPTION

[0017] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0018] Secondly, the present invention is described in detail with reference to schematic diagrams. When describing the embodiments of the present invention in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0019] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0020] The present invention inoculates Schneider membrane mesenchymal stem cells (SMMSCs) on the surface of PRF and induces osteogenic differentiation in this biological system, so that SMMSCs differentiate into osteoblasts in the natural ecological niche provided by PRF, secrete osteogenic-related proteins and bone matrix, and gradually wrap and replace PRF over time to obtain a bone tissue engineering material with sustained biological activity. Please refer to Fig. 9 , the specific implementation is as follows: 1. SMMSCs Isolation, Identification and Culture Previous studies have shown that there are two different types of mesenchymal stem cells in the periosteum and lamina propria of the human maxillary sinus mucosa, namely Schneider's membrane, and they are named maxillary sinus mucosa periosteum-derived mesenchymal stem cells (PMSCs) and lamina propria-derived mesenchymal stem cells (LMSCs). The present invention uses two types of SMMSCs to co-culture with PRF to obtain corresponding bone graft materials and detect their related properties.

[0021] 1) Cell acquisition The obtained healthy human maxillary sinus mucosal tissue of about 1×1 cm was immersed in sterile phosphate buffered saline (PBS) containing 10% penicillin-streptomycin, temporarily stored on ice, and subsequently processed within 2 h. The periosteum layer of the maxillary sinus mucosa was torn off and separated into tissue blocks no larger than 0.3×0.3 cm and placed in a T25 culture flask for in vitro culture of PMSCs. The remaining maxillary sinus mucosal tissue was placed in 2 ml of dispase neutral protease at 37°C to digest and remove the epithelial layer. The remaining periosteum layer was washed with PBS and separated into tissue blocks no larger than 0.3×0.3 cm and placed in a T25 culture flask for in vitro culture of LMSCs. The complete culture medium consisted of α-MEM medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. The culture conditions were 37°C, 5% CO 2 The medium was fully replaced every 2-3 days until the cell confluence reached 80%-90% for subculture.

[0022] 2) Passaging The cells were digested with 0.25% trypsin solution and terminated with twice the volume of culture medium containing 10% fetal bovine serum. The cells were collected by centrifugation at 300g for 5min and inoculated into new culture flasks according to the cell confluence of 50%-60%.

[0023] 3) Cryopreservation and thawing When freezing, after cell digestion and centrifugation, remove the supernatant and add cell freezing solution with a ratio of 92% dimethyl sulfoxide + 8% fetal bovine serum. Use the gradient freezing method to preserve cells, the method is 4℃, 30min; -20℃, 30min; -80℃, overnight; liquid nitrogen, long-term storage. When reviving, take out the cells and quickly put them in a 37℃ water bath until the liquid melts, add 9 times the volume of complete culture medium and mix well, then centrifuge at 300g for 5min to collect the cells, and inoculate them into new culture bottles according to the cell confluence of 60%-70%.

[0024] 4) Identification of mesenchymal stem cells: Flow cytometry was used to detect cell surface markers, including CD73, CD90, CD105, CD34, CD45 and HLA-DR. The operation method was to prepare a cell suspension and adjust the density to 1×10 6 Place 100 μl of cells / 1.5 ml EP tubes, add 1 μl of flow cytometry labeled antibody to each tube and incubate at 4°C in the dark for 30 min. Tap the tube wall every 5 min to ensure uniform antibody binding. Then collect the cells by centrifugation at 300 g for 5 min, wash twice with PBS containing 0.5% BSA and resuspend the cells to 500 μl for detection.

[0025] The osteogenic, adipogenic, and chondrogenic differentiation kits of human stem cells (Saiye (Guangzhou) Biotechnology Co., Ltd.) were used to induce and identify the three differentiations of SMMSCs. The specific methods were carried out according to the instructions provided by the manufacturer.

[0026] 2. Preparation of PRF membrane 10 ml of blood was drawn from the elbow vein and placed in a sterile thrombin-free blood collection tube; the tube was immediately centrifuged at 3000 rpm for 10 min; after standing, three layers appeared in the tube, from the bottom to the top, namely the red blood cell layer, the white gel-like PRF layer, and the platelet-poor serum layer. The middle PRF layer was clamped with sterile tweezers and placed on the surface of three layers of sterile gauze, covered with three layers of gauze and pressed gently and slowly to remove moisture from the PRF gel block to obtain a PRF membrane sheet.

[0027] Co-culture of SMMSCs and PRF One PRF membrane sheet obtained from 10 ml of venous blood was placed in a 24-well plate and 1 × 105 LMSCs or PMSCs were inoculated on the surface of a PRF membrane, and the complete culture medium was α-MEM medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. After 24 hours of co-culture, it was replaced with osteogenic induction medium, which was a complete culture medium containing 0.01 M β-glycerophosphate sodium + 0.25 mM ascorbic acid + 100 nM dexamethasone, and co-culture was continued until the 7th day for use, during which the osteogenic induction medium was fully replaced every 2 days.

[0028] Heterotopic transplantation Male 6-week-old BALB / c-nu nude mice weighing 20 g were used for in vivo transplantation experiments. Animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee and in strict compliance with the ethical principles of experimental animal welfare (CKARI202317). PRF loaded with LMSCs or PMSCs cultured to day 7 were stained with Dil before implantation to mark the cells loaded on the surface of PRF. Before surgery, nude mice were anesthetized, 1.5% sodium pentobarbital (0.1 ml / 20 g) was injected intraperitoneally, a 3 mm incision was made on the back skin, the material was implanted subcutaneously, and the skin was tightly sutured. Penicillin sodium was injected intramuscularly for 3 days after surgery. All nude mice recovered well after surgery, were housed in single cages and provided with adequate food and water. One week after surgery, the nude mice were euthanized under deep anesthesia. The transplanted tissues and some surrounding host tissues were removed and fixed with 4% paraformaldehyde (PFA, PH=7.4) for 24 h before subsequent testing.

[0029] Hematoxylin and eosin (H&E) staining After fixing the tissue samples in 4% PFA, they were dehydrated in gradient ethanol (80%-90%-95% ǀ-95% ǁ-100% ǀ-100% ǁ-100% Ⅲ), transparentized with xylene, and embedded in paraffin to make 3µm sections. Before H&E staining, the sections were dewaxed and hydrated, then stained in eosin stain and hematoxylin stain in sequence. After staining with each stain, the sections were washed with running water to remove excess dye. After staining, they were sequentially dehydrated, transparentized, and sealed with neutral gum for observation under a microscope.

[0030] Tissue immunofluorescence staining The tissue samples were prepared into 3µm sections and dewaxed and hydrated as above. Antigen retrieval of the tissue was performed at 37°C using pepsin retrieval solution. After 30 minutes, the tissue sections were washed with TBS buffer containing 1% w / v Triton X-100. Nonspecific binding was blocked using immunostaining blocking solution. Primary antibodies were added and incubated overnight at 4°C. Antibody indicators were: rabbit anti-human VEGF and OCN. The sections were washed the next day, and the tissue sections were incubated at room temperature for 60 minutes using FITC goat anti-rabbit IgG (H+L) and washed. The specimens were sealed using anti-fluorescence quenching sealing solution containing DAPI. Images were acquired using a fluorescence microscope.

[0031] Single-cell sequencing The cells in PRF loaded with LMSCs or PMSCs and cultured to the 7th day were fully digested with 0.25% trypsin and collected by centrifugation. The quality of the single cell suspension was checked and counted, and the cell survival rate was above 90%. The qualified cells were washed and resuspended to prepare 700-1200 cells / ul for operation on the 10x Genomics Chromium™ system. GEMs (gel beads in emulsion) were constructed for single cell isolation. After the GEMs were formed normally, the GEMs were collected and reverse transcribed in a PCR instrument for labeling. The GEMs were broken and the first-strand cDNA was purified and enriched with magnetic beads, and then cDNA was amplified and quality checked. The 3ʹ-end gene expression library was constructed with qualified cDNA. After fragmentation, adapter ligation, sample index PCR and other operations, the library was finally quantitatively detected using Qubit. Sequencing was performed using the Illumina NovaSeq 6000 sequencing platform in PE150 sequencing mode.

[0032] The data from the Illumina platform were used for bioinformatics analysis. All analyses were completed by Shanghai Zhongkexin Life Science Co., Ltd. The main process is as follows: 1) Sequencing data quality control The raw sequencing data was analyzed using FastQC software to calculate the quality value of each base and check the A / T / G / C content distribution.

[0033] 2) CellRange analysis Cell Ranger software was used to perform data quality statistics on the original Fastq data, and STAR was used to align the read2 sequence to the reference genome of the Ensembl database (https: / / asia.ensembl.org / index.html). The barcode sequences in the column used to distinguish cells and the UMI markers of different mRNA molecules in each cell are used, and the average of the 50% barcodes with the least UMI content is used as the empty cell. The transcription profile of each remaining barcode is checked to see if it is similar to the empty cell, so as to quantify the high-throughput single-cell transcriptome results. Based on the expression matrix generated by Cell Ranger, the Seurat package is used for cell filtering, and outliers (multiple cells, dead cells, empty cells, etc.) are removed based on the number of genes (Genes), UMI number (UMIs), mitochondrial gene expression, ribosomal gene expression and other information expressed in each cell.

[0034] 3) Cell clustering analysis Cluster analysis was performed using the Seurat package. First, the PCA method was used to perform moderate dimensionality reduction to reduce the amount of calculation and noise; then the Leiden method was used to find the modules of the neighboring cell network in the dimensionality reduction space; finally, the UMAP nonlinear dimensionality reduction method was used to visualize the clustering of single-cell groups. When it comes to multi-sample integrated analysis, their difference vectors are calculated through Seurat, and then the gene expression values ​​of the anchor cells are corrected to eliminate technical bias, realize the integration of multiple single-cell data sets, and finally perform cell clustering.

[0035] 4) Cell marker gene analysis Seurat package was used, and Wilcoxon test was adopted to select the significant difference range of FDR≤0.05 and |Log2FC|≥1.5. Screen the specific differentially expressed genes between the designated cell population and the rest of the cell populations, and the top-ranked differentially expressed genes can be used better It is a candidate gene for marker gene.

[0036] 5) Cell type prediction SingleR software was used to annotate cell types in combination with the Human Primary Cell Atlas Data database. After defining the cell types, the number and proportion of cell types in each sample were counted.

[0037] Depend on Figure 1-Figure 8 It can be seen that the present invention uses two types of human Schneider membrane mesenchymal stem cells LMSCs and PMSCs as seed cells for tissue engineering. The cells grow well under light microscopy, and all show spindle-shaped fibroblast morphology, with occasional short protrusions in the cell body. The cell body of LMSCs is relatively short, while that of PMSCs is relatively slender ( Figure 1). LMSCs and PMSCs were identified as mesenchymal stem cells (MSCs). The results showed that both cells can differentiate into osteoblasts, adipocytes, and chondrocytes ( Figure 2 ), and clearly expressed MSCs positive markers CD73, CD90 and CD105, and did not express MSCs negative markers CD34, CD45 and HLA-DR ( Figure 3 ). This provides a biological basis for the successful preparation of the material. PRF gel was prepared and pressed into PRF membrane. The PRF membrane after dehydration was white and translucent ( Figure 4 ). After LMSCs and PMSCs were inoculated into PRF, H&E staining of histological sections clearly showed that the surface of PRF was loaded with evenly and densely arranged cells. It was also observed that in an environment where MSCs continuously secreted biological enzymes, as PRF degraded, LMSCs and PMSCs gradually grew into the area that replaced the original fibrin of PRF, forming a denser tissue-like structure ( Figure 5 ). The above results show that both LMSCs and PMSCs can be well attached to the surface of PRF and have a good growth trend, which helps them to exert stable biological functions. The results of tissue immunofluorescence staining show that PRF loaded with LMSCs and PMSCs can significantly express angiogenesis-related protein VEGF and osteoblast-related protein OCN. In particular, PRF loaded with LMSCs expressed VEGF more significantly, and PRF loaded with PMSCs expressed OCN more significantly ( Figure 6 ). The results of heterotopic transplantation in nude mice also showed that VEGF and OCN were clearly expressed in the area where LMSCs and PMSCs were previously labeled in vitro, and the trend was consistent with the in vitro results ( Figure 7 ). Single-cell sequencing results showed that the cell types separated from the PRF materials loaded with LMSCs and PMSCs mainly included mesenchymal stem cells, osteoblasts and fibroblasts. In addition, there were also a small number of B cells, T cells, white blood cells and platelets derived from PRF. The proportion of fibroblasts (which can induce angiogenesis) in the PRF loaded with LMSCs was slightly higher, and the proportion of osteoblasts in the PRF loaded with PMSCs was slightly higher ( Figure 8 ). Combined with the above results, it is shown that PRF loaded with LMSCs and PMSCs tends to be a bioactive material for inducing angiogenesis and osteogenesis, respectively.

[0038] Although the present invention has been described above with reference to the embodiments, various modifications may be made thereto and parts thereof may be replaced by equivalents without departing from the scope of the present invention. In particular, as long as there is no structural conflict, the various features in the embodiments disclosed in the present invention may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A bioactive material for inducing angiogenesis and osteogenesis, characterized in that: include: Schneider membrane mesenchymal stem cells and platelet-rich fibrin membrane sheet, wherein the Schneider membrane mesenchymal stem cells are loaded on the surface of the platelet-rich fibrin membrane sheet.

2. The bioactive material for inducing angiogenesis and osteogenesis according to claim 1, characterized in that: The Schneider membrane mesenchymal stem cells include mesenchymal stem cells derived from the mucoperiosteum layer of the maxillary sinus and mesenchymal stem cells derived from the lamina propria.

3. A method for preparing the bioactive material for inducing angiogenesis and osteogenesis as claimed in claim 1 or 2, characterized in that: Here are the steps: S1. Place a platelet-rich fibrin membrane obtained from 10 ml of venous blood in a 24-well plate and use complete culture medium to culture 1×10 5 Schneider membrane mesenchymal stem cells were seeded on the surface of a platelet-rich fibrin membrane, and the complete culture medium was α-MEM medium containing 10% fetal bovine serum + 1% penicillin-streptomycin; S2. After 24 h of co-culture, replace the medium with osteogenic induction medium, which is a complete medium containing 0.01 M β-sodium glycerophosphate + 0.25 mM ascorbic acid + 100 nM dexamethasone. Continue co-culture until the 7th day for use. During this period, the osteogenic induction medium is fully replaced every 2 days.

4. The method of inducing angiogenesis and osteogenesis of bioactive materials according to claim 1, characterized in that: In step S1, Schneider membrane mesenchymal stem cells are obtained by the following steps: The obtained 1×1 cm healthy human maxillary sinus mucosal tissue was immersed in sterile phosphate buffer containing 10% penicillin-streptomycin, stored on ice, and subsequently processed within 2 h; The periosteum layer of the maxillary sinus mucosa was torn off and separated into tissue blocks no larger than 0.3×0.3 cm and placed in a T25 culture flask for in vitro culture of PMSCs. The remaining maxillary sinus mucosa tissue was placed in 2 ml of dispase neutral protease at 37°C to digest and remove the epithelial layer. The remaining periosteum layer was washed with PBS and separated into tissue blocks no larger than 0.3×0.3 cm and placed in a T25 culture flask for in vitro culture of LMSCs. The complete culture medium consists of α-MEM medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. The culture conditions are 37°C, 5% CO2. The medium is replaced every 2-3 days until the cell confluence reaches 80%-90% for passage. The cells were digested with 0.25% trypsin solution and terminated with twice the volume of culture medium containing 10% fetal bovine serum. The cells were collected by centrifugation at 300 g for 5 min and inoculated into new culture flasks according to the cell confluence of 50%-60%; Then cryopreservation is carried out. During cryopreservation, the cells are digested and centrifuged, the supernatant is removed, and cell freezing solution is added, with a ratio of 92% dimethyl sulfoxide + 8% fetal bovine serum. The cells are preserved using the gradient freezing method. When reviving, the cells are taken out and quickly placed in a 37°C water bath until the liquid melts. After adding 9 times the volume of complete culture medium and mixing evenly, the cells are collected by centrifugation at 300g for 5 minutes and inoculated into new culture bottles according to the cell confluence of 60%-70%.

5. The method of inducing angiogenesis and osteogenesis of bioactive materials according to claim 1, characterized in that: In step S1, the platelet-rich fibrin membrane is obtained by the following steps: 10 ml of blood was drawn from the elbow vein and placed in a sterile thrombin-free blood collection tube, which was immediately centrifuged at 3000 rpm for 10 min. After standing, three layers appeared in the tube, from the bottom to the top, namely the red blood cell layer, the white gel-like PRF layer, and the platelet-poor serum layer. Use sterile tweezers to pinch the middle PRF layer and place it on the surface of three layers of sterile gauze. Cover the three layers of gauze and press gently and slowly to remove moisture from the PRF gel block to obtain a platelet-rich fibrin membrane sheet.

6. A bioactive material for inducing angiogenesis and osteogenesis in the field of bone regeneration, characterized in that: Serves as a barrier membrane to guide tissue regeneration.

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