CN111282020A - Matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and preparation method thereof - Google Patents

Abstract

The invention discloses a method for preparing a matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells. The mesenchymal stem cells and endothelial progenitor cells are planted on a porous bone scaffold material in a ratio of 1:1 The tissue engineering complex was constructed, and the osteogenic induction medium was added in vitro to continue culturing for 13 to 15 days, frozen at -70 to 80°C for 48 to 72 hours, freeze-dried for 24 to 30 hours, and cryopreserved for 1 to 3 months. Matrix-dependent tissue-engineered bone is obtained. The results of in vitro studies show that the matrix-dependent tissue engineered bone based on mesenchymal stem cells/endothelial progenitor cells as seed cells constructed by the method of the present invention can significantly promote endothelial progenitor cell migration, scratch repair and lumen formation in vitro, as well as promote Osteogenic differentiation of mesenchymal stem cells; in vivo experiments show that they can significantly promote new bone formation and successfully repair bone defects.

Description

Matrix-dependent construction based on mesenchymal stem cells/endothelial progenitor cells as seed cells Lay-type tissue engineered bone and preparation method thereof

technical field

The invention belongs to the technical field of tissue engineering, and relates to a matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and a preparation method thereof.

Background technique

The treatment of large segmental bone defects caused by trauma, tumor and infection has always been one of the difficulties in clinical treatment. Autologous bone transplantation is still the "gold standard" for clinical bone transplantation, but there are limitations such as less bone harvesting and trauma to the bone harvesting area. Tissue engineering bone (TEB) is a hot research topic in recent years, and has achieved good clinical results, but there are also problems such as dependence on living cells, dependence on autologous cells, and harsh storage and transportation conditions.

In order to solve the above problems, we previously established a bone matrix material containing various proteins secreted by umbilical cord mesenchymal stem cells and its preparation method (Patent ZL 2013 1 0449152.5), and on this basis, proposed a "matrix-dependent tissue engineered bone matrix". (Extracellular matrix-based tissue engineering bone, ECM-TEB)" construction concept and system. That is, ECM-TEB is constructed and obtained by removing the activity of seed cells and retaining active proteins such as various cytokines secreted by cells. Although this technical method removes cell activity, the cytokines and matrix proteins that the cells are wrapped on the scaffold material layer by layer in an autocrine manner are still retained. After transplantation in vivo, the cytokines wrapped in the matrix under the action of enzymes are slowly released to damage Therefore, the bioactive proteins loaded and released by the decellularized ECM-TEB meet the needs of the physiological environment. The basic construction strategy is as follows: mesenchymal stem cells (MSCs) are composited with bone scaffolds for 14 days, and the cell-scaffold composites are frozen at -80°C for 48 hours, freeze-dried for 24 hours, and cryopreserved for 3 days. months to significantly reduce immunogenicity to form MSCs as seed cells for ECM-TEB. This strategy retains 80% of the active protein, which effectively overcomes the long time and high cost of individualized tissue engineering of bone, and is affected by the state of the patient's autologous stem cells. Research provides new ideas.

The dynamic balance of bone remodeling is achieved by the cooperation of various types of cells in the bone tissue. Therefore, if different types of cells can be established for co-culture in the construction of ECM-TEB, the microenvironment of in vivo osteogenesis will be more realistically simulated. The MSC ECM-TEB constructed with MSC as a single seed cell shows a good application prospect in repairing small-sized bone defects, but when repairing large bone defects, due to the lack of a perfect vascular system, blood vessels at the defect site are often caused. The chemical difference limits the clinical application of tissue-engineered bone. With the development of regenerative medicine, ECM-TEB with angiogenic potential is of great significance for repairing large segmental bone defects. Endothelial progenitor cells (EPC) can differentiate into vascular endothelial cells to generate blood vessels, and can promote the homing and osteogenesis of MSCs in a specific microenvironment. Therefore, this is the use of MSCs as seed cells for osteogenic differentiation in the present invention. At the same time, it is of great clinical significance to introduce EPC as seed cells to explore the MSC/EPC matrix-dependent tissue engineered bone constructed by the "composite" seed cells.

At present, there has not been any report on the use of endothelial progenitor cells as one of the seed cells for matrix-dependent tissue engineering bone construction and the treatment of bone defects.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to establish a new type of tissue-engineered bone to construct a new type of tissue-engineered bone when currently using bulk tissue-engineered bone to repair large-segment bone defects, and to introduce EPC as one of the seed cells, so as to solve the problem of only using MSC as a single seed cell. The source to construct matrix-dependent tissue-engineered bone has the problem that it is difficult to achieve a more realistic biomimetic in vivo osteogenic microenvironment, and it is easy to lead to the problem of poor vascularization at the defect site. The present invention provides the following technical solutions:

1. The preparation method of matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells, the mesenchymal stem cells and endothelial progenitor cells are planted on the porous bone scaffold material at a ratio of 1:1 to construct a tissue The engineering complex was then added to the osteogenic induction medium in vitro for 13 to 15 days, frozen at -70 to 80°C for 48 to 72 hours, freeze-dried for 24 to 30 hours, and cryopreserved for 1 to 3 months to obtain the matrix. Dependent tissue engineered bone.

Further, the mesenchymal stem cells are bone marrow mesenchymal stem cells, peripheral blood mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, adipose mesenchymal stem cells or umbilical cord mesenchymal stem cells.

Further, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

Further, the porous bone scaffold material is a synthetic bone scaffold material, acellular bone matrix or demineralized bone matrix.

Further, the porous bone scaffold material is demineralized bone matrix.

Further, the preparation steps are: taking the demineralized bone matrix scaffold material, soaking it in culture medium for 48 hours, and adjusting the pH value to 7.2; taking the second generation of mesenchymal stem cells in the exponential growth phase, washing with PBS, and adding 0.1 wt% Incubate with collagenase type I solution for 1 hour, then add trypsin and 0.02wt% EDTA solution for 3-5 minutes, wash and resuspend in DMEM medium containing 10wt% fetal bovine serum, and carry out 1:1 with endothelial progenitor cells Mix and resuspend; inoculate the cells on the decalcified bone matrix scaffold material after the aforementioned treatment, so that the cell suspension just infiltrates the scaffold material and does not overflow the scaffold material; after 3 hours, the decalcified bone matrix is placed under sterile conditions. The bottom surface of the scaffold material is turned into the top surface, and the cell suspension is inoculated with the aforementioned method; the cell scaffold is 0.5cm×0.5cm×0.3cm in regular size, and the total number of planted cells is about 10 ⁶ ; DMEM medium is added after 3 hours to just not overgrown. The top surface of the calcium-bone matrix scaffold material was cultured at 37°C and 5% CO ₂ ; after 24 hours, it was cultured in vitro for osteogenic differentiation induction for 13-15 days; frozen at -70-80°C for 48- 72 hours, freeze-drying for 24-30 hours, cryopreservation for 1-3 months to significantly reduce immunogenicity, and form matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells.

2. A matrix-dependent tissue engineered bone constructed by using the above preparation method based on mesenchymal stem cells/endothelial progenitor cells as seed cells.

Beneficial effects of the present invention: The present invention establishes for the first time a matrix-dependent tissue engineered bone based on mesenchymal stem cells/endothelial progenitor cells as seed cells, which not only broadens the construction method of matrix-dependent tissue engineered bone, but also improves A good solution to the vascularized biomimetic microenvironment. The results of in vivo studies show that the matrix-dependent tissue engineered bone constructed by the method of the present invention based on mesenchymal stem cells/endothelial progenitor cells as seed cells can successfully repair bone defects in vivo, in terms of cell recruitment, angiogenesis and new bone formation. The advantages are significantly enhanced, and the problems such as the vascular microenvironment of the bone graft bed are effectively solved, so the invention has a good application prospect in tissue engineering bone construction and bone defect repair.

Description of drawings

In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the following drawings are provided for description of the present invention.

Figure 1 shows the construction strategy of matrix-dependent tissue engineered bone based on mesenchymal stem cells/endothelial progenitor cells as seed cells.

Figure 2 is a graph showing the results of in vitro scratch repair experiments on EPC by protein extracts of matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells.

Figure 3 is a graph showing the results of in vitro migration experiments on EPCs from matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells.

Fig. 4 is a graph showing the results of an experiment on the osteogenic differentiation of MSCs by protein extracts of matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells.

Figure 5 is a graph showing the experimental results of in vitro lumen formation of EPCs by protein extracts of matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells.

Figure 6 shows the results of the experiment of repairing rat femoral defects with matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells, and the results of Micro-CT examination and Masson staining 2 months after implantation.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. In the preferred embodiment, the experimental methods without specific conditions are usually carried out according to conventional conditions, or according to the conditions suggested by the reagent manufacturer.

Example 1. Construction of matrix-dependent tissue engineered bone based on mesenchymal stem cells/endothelial progenitor cells as seed cells

1. Culture of endothelial progenitor cells

Take a 15mL centrifuge tube, add 5mL bone marrow lymphocyte separation solution, carefully aspirate the bone marrow single cell suspension and add it to the surface of the separation solution, centrifuge at 400-500g for 30min at low temperature. Carefully aspirate the second layer of lymphocytes, wash the cells with washing solution, inoculate in a Petri dish lined with Matrigel, add EGM2-MV medium for culture, and replace with fresh medium for the first time after 3 days. After 7 days of cell culture, the cells were washed twice with PBS, Dil-ac-LDL (10 μg/mL) was added, and the adherent cells were incubated at 37°C in the dark for 4 hours, washed three times with PBS, and fixed with 5% paraformaldehyde for 15-20 min. After washing 3 times with PBS, FITC-UEA-1 (10 μg/mL) was added and incubated in a 37°C incubator for 1 hour. After rinsing the cells 3 times with PBS, the cells uptake of Dil-ac-LDL and Dil-ac-LDL were observed under a fluorescence microscope. Identification was carried out in combination with FITC-UEA-1 capacity. EPCs primitive cells were round, adhered within 72 hours, and were mainly spindle-shaped. The double-cytosis experiment identified DIL-AC-LDL and FITC-UEA-1 double positive cells as endothelial progenitor cells.

2. Culture of umbilical cord MSCs

Take the healthy fetal umbilical cord, cut off the arteries and veins, cut into pieces; use tissue block culture, digest the umbilical cord with type I collagenase; wash the tissue block with phosphate buffer, transfer the tissue block into a culture bottle, add medium for adherent culture; Culture in a 37°C, 5% CO2 constant temperature incubator, change the medium every 3 days, observe the cell growth and morphological characteristics under an inverted microscope after changing the medium, digest with 0.25% trypsin when the cells are close to confluence, and use 1:2 Proportional passaging. When the bone marrow MSCs cultured above were confluent to about 80%, they were digested with a 1:1 mixture of 2.5g/L trypsin and 0.2g/L EDTA, and seeded in a subculture flask (T25) at a cell density of 8.0×10 ³ /cm2. ), and the cell growth and morphological characteristics were observed under an inverted microscope. The results showed that the cells of the second generation grew well and had a uniform shape, and most of the cells were mature mesenchymal stem cells with broad, large or flat shape.

3. Construction of matrix-dependent tissue engineered bone based on mesenchymal stem cells/endothelial progenitor cells as seed cells

The construction strategy is shown in Figure 1. Demineralized bone matrix (DBM) scaffold material (volume 0.5cm×0.5cm×0.3cm) was taken, placed in a 6-well plate, soaked in DMEM medium for 48 hours, and adjusted to a pH value of about 7.2; The second generation MSCs in the exponential growth phase were taken, washed twice with PBS, incubated with 0.1% collagen type I (Col I) enzyme for 1 h, then added with 0.25% trypsin and 0.02% EDTA to digest for 3-5 min, and cultured with DMEM. The basal cells were washed and resuspended, mixed with endothelial progenitor cells at a ratio of 1:1 and resuspended, and the total number of seeded cells was 10 ⁷ . The cells are seeded on the demineralized bone matrix scaffold material after the aforementioned treatment, so that the cell suspension does not overflow the scaffold material. After 3 hours, the bottom surface of the demineralized bone matrix scaffold was turned over to the top surface under sterile operating conditions, and the cell suspension was seeded in the same way as described above. After 3 hours, DMEM medium was added to just cover the top surface of the demineralized bone matrix scaffold material, and then cultured at 37° C. and 5% CO ₂ . After 24 hours, they were cultured in vitro for osteogenic differentiation induction for 13 to 15 days. The osteogenic induction medium was: DMEM(H) + 10% FBS + 10 mmol/L β-sodium glycerophosphate + 0.1 μmol/L dexamethasone + 50 mg/LVitC. Scanning electron microscope observation showed that the cells were successfully seeded on the scaffold material. It is then frozen at -70 to 80°C for 48 to 72 hours, freeze-dried for 24 to 30 hours, and cryopreserved for 1 to 3 months to significantly reduce immunogenicity, forming osteoclast precursors and mesenchymal stem cells as seed cells. Matrix-dependent tissue engineered bone.

Example 2. In vitro migration, repair and osteogenic differentiation detection of matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells

1. In vitro scratch repair experiment: Inoculate endothelial cells (10 ⁶ cells/well) on a 6-well plate and continue to incubate at 37°C. When the cells reach 90% growth density, scrape them with a 200 μl pipette tip. Wipe the cell surface, then wash the cells with PBS to remove debris, and use the matrix-dependent tissue engineered bone (MSC/EPC group) constructed by the present invention based on mesenchymal stem cells/endothelial progenitor cells as seed cells, and pure MSC as the seed cells. The matrix-dependent tissue engineered bone of seed cells (MSC group) was further cultured with the two protein extracts and the medium containing 2% serum in a ratio of 1:1. Changes in the scratched area were observed and observed with a microscope at t=0 and 12 hours and the size of the scratched area was measured with Image J. Scratch healing rate (%)=(A0-A6)/A0×100%, where A0 represents the initial scratch area area (t=0 hours) and A6 represents the final scratch area area at t=6 hours. As shown in Figure 2, A and B are the scratch repair conditions of the MSC group and the MSC/EPC group at the beginning (t0), respectively; C and D are the scratches of the MSC group and the MSC/EPC group after 6 hours (t6), respectively. The rate of tissue repair in the MSC/EPC group was (61.49±5.85)%, and that in the MSC group was (39.34±3.61)%, and the MSC/EPC group was significantly better than the control MSC group (t=5.581, P<0.01 ).

2. In vitro migration experiment: The cell migration experiment was carried out using a 24-well plate. Bone marrow MSCs were planted in the upper chamber of the Transwell culture system at 500 μL of 10 ⁶ cells/mL, and the lower chamber was the mesenchymal stem cells/endothelial progenitor constructed by the present invention. The cells were used as seed cells to construct matrix-dependent tissue-engineered bone (MSC/EPC group), and the matrix-dependent tissue-engineered bone with pure MSCs as seed cells (MSC group) was used as a control. After 24 hours, cells that migrated to the underside of the filter were fixed with 4% paraformaldehyde and stained with DAPI. As shown in Figure 3, A. MSC group cells migrated by DAPI staining; B. MSC/EPC group cells migrated by DAPI staining. The results showed that the number of cells migrated in the MSC/EPC group was (121.63±8.30), and the MSC group was (84.97±6.68), and the MSC/EPC group was significantly better than the control MSC group (t=5.96, P<0.01).

3. In vitro osteogenic differentiation experiment: when the second-generation bone marrow MSCs reached a growth density of 80% to 90%, they were digested with trypsin for ⁵ minutes, centrifuged at 1000 r/min for 3 minutes, and the cell suspension was prepared in F12 medium. Each well was seeded in a 24-well plate, and the matrix-dependent tissue engineered bone constructed by the present invention based on mesenchymal stem cells/endothelial progenitor cells as seed cells (MSC/EPC group) and pure MSC were used as the matrix of seed cells. Dependent tissue-engineered bone (MSC group) was cultured in a 1:1 ratio of these two protein extracts and medium containing 10% serum. Alizarin red staining and ALP staining were performed after 21 days, and observed under an inverted microscope. Calcium nodules were formed and grossly photographed. The results are shown in Fig. 4, A and B in Fig. 4 are the alizarin red staining of the MSC group and the MSC/EPC group, respectively; C and D in Fig. 4 are the ALP staining of the MSC group and the MSC/EPC group, respectively. The results showed that the results of evaluating the osteogenic differentiation ability of the two matrix-dependent tissue engineered bones by alizarin red staining and ALP staining were basically the same. The mineralization ratio of the MSC group was about 38%, and the mineralization rate of the MSC/EPC group The ratio is 45% to 50%, with obvious differences.

4. In vitro lumen formation experiment: The second generation EPCs were seeded at 5×10 ⁴ /well in a 24-well plate plated with 200uL/well Matrigel, and the cells based on mesenchymal stem cells/endothelial progenitor cells were used as seed cells respectively. The constructed matrix-dependent tissue-engineered bone (MSC/EPC group) and the matrix-dependent tissue-engineered bone with pure MSCs as seed cells (MSC group) two protein extracts and 10% serum-containing culture medium in a ratio of 1:1 The mixed culture medium was incubated for 6 hours, and the formation of the lumen was observed using an inverted microscope, and 5 fields of view were randomly selected to take pictures. The results are shown in Figure 5. Figure 5A shows the lumen formation in the MSC group, and Figure 5B shows the lumen formation in the MSC/EPC group. It can be seen from Figure 5 that the length of the lumen formation in the MSC/EPC group is (11.31±0.59 ) mm, the MSC group was (5.90±0.36)% mm, and the MSC/EPC group was significantly better than the control MSC/EPC group (t=13.56, P<0.001).

Example 3. Repair of bone defects in matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells

Animal model preparation and experimental grouping: Healthy 2-month-old SD rats with a body weight of (200±20) g were used to expose bilateral femoral condyles under sterile conditions after anesthesia, and dental drills were used to create bone defects with a diameter of 3 mm. Rats received the following three groups of implants to repair bone defects: (1) blank control group: only debridement was performed on the defect; (2) control group: matrix-dependent tissue engineered bone with only MSCs as seed cells; (3) Experimental group: the matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells of the present invention. The defect was washed with hydrogen peroxide, iodophor and normal saline repeatedly, and sutured in layers. After the experimental animals recovered, they were kept in separate cages and injected with penicillin-streptomycin to prevent infection. After 2 months after operation, Micro-CT examination was performed, according to bone volume fraction (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N) and trabecular bone separation (Tb.Sp) ) to assess bone defect repair. Masson staining was performed for histological observation to evaluate the formation of new bone in the defect. The results of Micro-CT examination and Masson staining after 2 months of implantation are shown in Figure 6. A to C in Figure 6 are the Micro-CT images of the femoral defect in the blank control group, the control group and the experimental group 2 months after the operation; D to F in Figure 6 are the Masson staining histological images of the femoral defect in the blank control group, the control group and the experimental group 2 months after the operation. The results showed that after 2 months of operation on the femoral defect of the rat, there was almost no new bone formation in the blank control group, and a little new bone formation in the control group, but no complete bone structure appeared in the defect; the defect repair in the experimental group was basically completed, and the trabecular structure was basically completed. Basically complete. These results suggest that matrix-dependent tissue-engineered bone in the MSC/EPC group has advantages in promoting osteogenesis. Micro-CT examination analysis of A to C in Figure 6 shows that the experimental group was significantly stronger than the experimental group in terms of bone volume fraction (BV/TV), trabecular bone number (Tb.N) and trabecular bone thickness (Tb.Th). The other two groups (P<0.01). Masson staining of D to F in Fig. 6 shows a large amount of new bone tissue, complete bone trabecular structure, and many osteoblasts and chondrocytes are seen locally. In the control group, there was a small amount of lamellar trabecular bone structure and a small amount of osteoblasts. In the blank control group, fibrous tissue structure was more common, and no obvious new bone was found.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should Various changes may be made in details without departing from the scope of the invention as defined by the claims.

Claims (7)

1. A method for preparing a matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells, characterized in that the mesenchymal stem cells and endothelial progenitor cells are planted on a porous bone scaffold at a ratio of 1:1 The tissue engineering complex was constructed on the material, and the osteogenic induction medium was added in vitro to continue to culture for 13 to 15 days, frozen at -70 to 80 °C for 48 to 72 hours, freeze-dried for 24 to 30 hours, and cryopreserved for 1 to 3 days. month, obtained matrix-dependent tissue-engineered bone. 2. The method for preparing a matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells according to claim 1, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells, Peripheral blood mesenchymal stem cells, cord blood mesenchymal stem cells, adipose mesenchymal stem cells or umbilical cord mesenchymal stem cells. 3 . The method for preparing a matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells according to claim 2 , wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells. 4 . 4. The method for preparing a matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells according to claim 1, wherein the porous bone scaffold material is an artificial synthetic bone scaffold material, Acellular bone matrix or demineralized bone matrix. 5 . The method for preparing matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells according to claim 4 , wherein the porous bone scaffold material is demineralized bone matrix. 6 . 6. The method for preparing a matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells according to any one of claims 1 to 5, wherein the demineralized bone matrix scaffold material is taken, Soak in medium for 48 hours, adjust the pH to 7.2; take the second generation of mesenchymal stem cells in the exponential growth phase, wash with PBS, incubate with 0.1wt% collagenase solution I for 1 hour, then add trypsin and 0.02wt% EDTA solution was digested for 3 to 5 minutes, washed with DMEM medium containing 10wt% fetal bovine serum and resuspended, mixed with endothelial progenitor cells at 1:1 and resuspended; the cells were inoculated into the decalcified cells after the aforementioned treatment On the bone matrix scaffold material, make the cell suspension just infiltrate the scaffold material and not overflow the scaffold material; after 3 hours, under aseptic operating conditions, turn the bottom surface of the demineralized bone matrix scaffold material into the top surface, and inoculate the same as the previous method. Cell suspension; the cell scaffold is 0.5cm×0.5cm×0.3cm in regular size, and the total number of seeded cells is about 10 ⁶ ; after 3 hours, add DMEM medium to just cover the top surface of the demineralized bone matrix scaffold material, and then at 37°C, Cultured under 5% CO ₂ conditions; 24 hours later, in vitro osteogenic differentiation induction culture for 13 to 15 days; frozen at -70 to 80°C for 48 to 72 hours, freeze-dried for 24 to 30 hours, and cryopreserved for 1 ~3 months to significantly reduce immunogenicity and form matrix-dependent tissue engineered bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells. 7 . A matrix-dependent tissue engineered bone constructed by using the preparation method according to any one of claims 1 to 6 based on mesenchymal stem cells/endothelial progenitor cells as seed cells. 8 .

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