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    • 专利摘要:
    The present invention discloses a matrix-dependent tissue-engineered bone having osteoclast precursors and mesenchymal stem cells as germ cells and a construction method thereof, wherein the mesenchymal stem cells and the osteoclast precursors are implanted at a ratio of 10:1 on a porous bone scaffold material to provide a tissue-engineering To construct complex, then an osteogenic induction culture medium is added in vitro, further culturing for 12-14 days, freezing for 48-72 hours at -70°C to -80°C, freeze-drying for 24-30 hours to induce matrix-dependent tissue to obtain engineering bones. The in vivo research results show that the present invention can promote new bone formation and successfully repair the bone defects.
    公开号:BE1028040B1
    申请号:E20215010
    申请日:2021-01-07
    公开日:2022-02-08
    发明作者:Jianmei Li；Mengmeng Liang；Rui Dong；wanyuan Liang；Jianzhong Xu；Fei Luo；Shiwu Dong；ce Dou；Yueqi Chen
    申请人:Univ Army Medical；
    IPC主号:
    专利说明:

    Matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells and construction methods therefor
    TECHNICAL FIELD The present invention relates to the technical field of tissue engineering and a matrix-dependent tissue engineering bone having osteoclast progenitors and mesenchymal stem cells as germ cells and a construction method thereof.
    BACKGROUND ART Treatment of large bone defects is always one of the difficult problems in clinical treatment. Autogenous bone is the "gold standard" for bone grafting, which, however, belongs to an invasive repair mode and cannot meet the enormous clinical need for bone repair materials. Afterwards, a variety of bone substitute materials are developed, such as e.g. B. xenogeneic bone, allogeneic decalcified bone and artificial bone substitute, etc. However, the presence of xenogeneic bone has the shortcomings that bone graft survival rate is low and it is prone to immune system rejection and disease spread, which is difficult to meet clinical needs can. The major shortcomings of allogeneic decalcified bones are that disease spread is easy to occur, a large bone is difficult to form in the recipient after transplantation, and fractures and infections are easy to occur, and there are also problems such as swelling. The development of tissue engineering over the past 20 years has opened up a new avenue for the repair and treatment of bone defects.
    The individualized tissue engineering bone can provide a large amount of high-activity bone repair materials for children and young patients, and has achieved good clinical results, providing good bone repair materials for patients with autogenous bone deficiency. The advantage is that only 10 mL of bone marrow should be taken from the patient, a large amount of germ cells are expanded in vitro and then inoculated on the scaffold material to construct the tissue engineered bone. After mature cultivation, a large amount of highly active bone repair materials can be obtained. The method is suitable for children and patients with high proliferative ability of stem cells in autogenous bone marrow, and its use on the elderly, especially patients with underlying diseases, is limited. Limitations include: 1) long construction time, requiring almost 3 weeks of in vitro amplification and cultivation; 2) high technical requirements, which requires specialized operating rooms with 100 levels and skilled workers; 3) high clinical cost, each case requires high cultivation cost on average. The core of the osteogenic activity of this conventional tissue engineered bone resides in the germ cells, but there are problems in the storage and transportation of the tissue engineered bone and the maintenance of the germ cell activity. In addition, many technical connections exist and quality control is difficult to achieve. It is used only in individual hospitals and individual groups. Large-scale clinical application and realization of industrialization are limited.
    The use of abundant proteins and factors in the extracellular matrix to repair tissue damage is one of the current research priorities. At an early stage, a bone matrix material containing a large amount of proteins secreted by umbilical cord mesenchymal stem cells and a manufacturing method (patent ZL 2013 1 0449152.5) were established, and based on this, a design concept and system for a matrix-dependent Tissue engineering bone "Matrix Based Tissue Engineering Bone, M-TEB)" developed. The basic construction strategy is as follows: the mesenchymal stem cells (MSC) are attached to skeleton materials, culturing for a total of 14 days, freezing the cell-scaffold complex for 48 hours at -80°C, and freeze-drying for 24 hours MSC to form ECM-TEB of germ cells. With this construction technology, the cytokines and
    Matrix proteins that wrap the cells autocrine onto the scaffold are preserved, although cellular activity is removed. After transplantation in vivo, the cytokines wrapped in the matrix are slowly released at the site of injury under the action of enzymes and take part in bone regeneration and reconstruction. Because of this, the biologically active protein loaded and released from the decellularized M-TEB satisfies the requirements of each physiological environment.
    Establishment of homeostasis and bone reconstruction in the bone microenvironment requires multiple cell interactions to perform physiological and pathological functions. Similarly, the dynamic balance of bone reconstruction is achieved through the cooperation of different cells in bone tissue. Therefore, if different cell types can be co-cultured in the construction of ECM-TEB, the microenvironment of bone formation in vivo will be more realistically simulated. The two ultimate functional cells required to maintain bone homeostasis are osteoblasts and osteoclasts. The osteoblasts (osteoblast, OB) originate from MSCs and are mainly responsible for the synthesis of the bone matrix and the deposition of calcium salts. The osteoclasts (osteoclasts, OC) are formed by cleavage of the monocyte/macrophage cell lineage in hematopoietic stem cells, and complete bone absorption and bone breakdown through local acid secretion and release of the bone matrix-degrading enzymes. The two cell types work closely together through precise regulation to complete the reconstruction of bone tissue and maintain bone homeostasis. The studies have shown that PDGF-BB secreted by osteoclast precursors (preosteoclasts, POC) can promote the mutual coupling of bone formation and vascularization, suggesting that osteoclast-related cells play a key role in bone repair and its vascularization. This provides a theoretical basis for the introduction of osteoclast precursors as "composite" germ cells in the present invention while using MSCSs as germ cells for osteogenic differentiation.
    At present there is no report on osteoclast progenitors as one of the germ cells for the construction of matrix-dependent tissue engineered bone and for the treatment of bone defects.
    SUMMARY OF THE PRESENT INVENTION In view of the above, the object of the present invention is to establish a novel tissue-engineered bone structure currently using a large tissue-engineered bone to repair large bone defects to solve the autogenous-living cell-dependency bottleneck problem Cells using the conventional tissue engineered bone and the problem that it is difficult to achieve a more realistic osteogenic microenvironment in vivo using MSC as a single source of germ cells. The present invention provides a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells and a construction method therefor. The present invention uses the following technical solution:
    1. A construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells, wherein the mesenchymal stem cells and the osteoclast precursors are implanted at a ratio of 10:1 on a porous bone scaffold material to construct a tissue engineering complex , then an osteogenic induction culture medium is added in vitro, further culturing for 12-14 days, freezing for 48-72 hours at -70°C to -80°C, freeze-drying for 24-30 hours to obtain a matrix-dependent tissue engineering bone to obtain.
    Preferably, the mesenchymal stem cells are bone marrow mesenchymal stem cells, peripheral blood mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, fat mesenchymal stem cells or umbilical cord mesenchymal stem cells.
    Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.
    Preferably, the porous skeletal material is a decellularized bone matrix or a decalcified bone matrix. Preferably, the porous skeletal material is a decalcified bone matrix.
    5 Preferably, take a scaffold with decalcified bone matrix, soak it for 48 hours in DMEM culture medium, adjust pH to 7.2; Take second generation mesenchymal stem cells in exponential growth phase, wash them with PBS and incubate for one hour using 0.1 wt% collagen enzyme solution of type L, then add 0.25 wt% pancreatin and 0.02 wt% % EDTA and digesting for 3-5 minutes, washing in DMEM culture medium containing 10% by weight fetal calf serum and resuspending, and mixing with the osteoclast precursors at a ratio of 10:1 and resuspending; Inoculating the cells onto the scaffold with decalcified bone matrix after the above treatment, so that the cell suspension only infiltrates the scaffold and does not overflow the scaffold, in 3 hours under aseptic operating conditions, the bottom of the scaffold with decalcified bone matrix is turned over to the top, and the cell suspension is inoculated using the procedure outlined above; the cell scaffold has a regular size of 0.5cm x 0.5cm x 0.3cm and the total number of cells implanted is about 10°, in 3 hours the DMEM culture substrate is added until the top of the scaffold material with decalcified bone matrix only slightly submerged, subsequent cultivation at 37°C and 5% CO». Performing induction cultivation for osteogenic differentiation in 24 hours in vitro for 12-14 days, freezing at -70°C to -80°C for 48-72 hours, freeze-drying for 24-30 hours and cryopreservation for 3 months to significantly increase the immunogenicity thus matrix-dependent tissue engineered bone is formed with osteoclast progenitors and mesenchymal stem cells as germ cells.
    The osteoclast precursors preferably originate from bone marrow monocytes, which are differentiated in the direction of the osteoclasts by 100 ng/mL RANKL and 50 ng/mL M-CSF for 24 hours.
    2. Using the above procedure, matrix-dependent tissue engineering bone is constructed with osteoclast precursors and mesenchymal stem cells as germ cells.
    The present invention has the following advantages: the present invention establishes for the first time a construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells, which not only expands the construction methods of the matrix-dependent tissue engineering bone, but also a more realistic one can construct osteogenic microenvironment in vivo. The results of in vivo studies show that the matrix-dependent tissue engineered bone constructed by the method of the present invention using osteoclast precursors and mesenchymal stem cells as germ cells can successfully repair the bone defects in vivo, so that the present invention in the construction of the tissue -Engineering bone and bone defect repair has a good application perspective.
    BRIEF DESCRIPTION OF THE DRAWING The present invention is explained in more detail in connection with the following figures, so that the aim, the technical solution and the advantages of the present invention become clearer.
    Figure 1 shows a schematic diagram of a strategy of a construction method for a matrix-dependent tissue engineering bone with osteoclast progenitors and mesenchymal stem cells as germ cells.
    Figure 2 shows a diagram of the detection result of a mixed lymphocyte culture reaction (MLR).
    Figure 3 shows a graph of the fluorescence detection results of MHC-I and MHC-II of a matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells.
    Figure 4 shows a graph of the results of an in vitro migration experiment of endothelial cells through a protein extract from matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells.
    Figure 5 shows a graph of the results of an MSC in vitro scratch repair experiment and in vitro osteogen differentiation experiment by a protein extract from a matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells.
    Figure 6 shows a graph of the results of an MSC in vitro osteogen differentiation experiment by a protein extract from a matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells.
    Figure 7 shows a graph of the micro-CT examination results of a matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells at 2 months after planting to repair femoral defects in rats.
    Figure 8 shows a graph of the results of Masson staining of matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells at 2 months after planting for repair of femoral defects in rats.
    DETAILED DESCRIPTION In connection with figures, the detailed embodiments of the present invention are explained in more detail below. In the preferred embodiments, the experimental procedures are not given in specific conditions, the experiments are usually performed according to conventional conditions or according to the conditions recommended by the reagent manufacturer.
    Example 1: Construction of a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells
    1. Culturing umbilical cord MSC Taking a healthy fetal umbilical cord, cutting off the arteries and veins and dissecting; culturing using a tissue block, digesting the umbilical cord with collagenase type I; washing the tissue block with a phosphate buffer solution, transferring the tissue block to a culture flask and adding the culture medium to perform wall-adherent cultivation; Culturing in a constant temperature incubator of 37°C and 5% CO», changing the substrate every 3 days, after changing the substrate, cell growth and morphological properties are observed under an inverted microscope when the cells are about to fuse, they are digested with 0.25% trypsin, and passed at a ratio of 1:2. When the above cultured bone marrow MSCs are approximately 80% fused, 2.5 g/L trypsin and 0.2 g/L EDTA are mixed at a 1:1 ratio and digested. The cells are grown in a subculture flask (T25) at a cell density of 8.0 x 10%/cm inoculated for propagation and cultivation. Cell growth and morphological characteristics are observed under an inverted microscope. The results show that the second generation cells grow well and have a uniform morphology. Most of the cells are mature mesenchymal stem cells in broad large polygons or flat shapes.
    2. Culturing Osteoclast Progenitors The primary bone marrow macrophages (BMMs) derived from bone marrow are used for induction culture. BMMs are seeded evenly into a cell culture plate (5000 cells/pore, 96-well plate), RANKL at a concentration of 100ng/mL and M-CSF at a concentration of 50ng/mL are used to induce cells to differentiate into osteoclasts . The induction time is 24 hours and the osteoclast precursors are preserved, which is evaluated and confirmed by the TRAP stain, the CFA stain, the fusion test and the bone phagocyte test. 80% of the cells at this stage are TRAP positive, but they are unable to absorb the bone.
    3. Construction of a matrix-dependent tissue-engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells The construction strategy is as shown in Figure 1, taking a scaffold material (cell scaffold 0.5cmx0.5cmx0.3cm) with decalcified bone matrix (DBM), Place in 6-well plate, soak in DMEM culture medium for 48 hours, adjust pH to about 7.2; Take the second generation of MSCs in the exponential
    growth phase, washing this with PBS twice and then incubating for 1 hour using 0.1% type I collagen enzyme (Col D, then adding 0.25% pancreatin and 0.02% EDTA and digesting for 3-5 minutes, wash in DMEM culture medium and resuspend, and mix with the osteoclast precursors at a ratio of 10:1 and perform resuspension Inoculate the cells onto the scaffold with decalcified bone matrix after the above treatment so that the cell suspension only infiltrates the scaffold and the scaffold material does not overflow.In 3 hours, under aseptic operation conditions, the bottom of the scaffold material is turned over to the top side with decalcified bone matrix, and the cell suspension is seeded by the above method, the total number of cells implanted is about 10°.In 3 hours, that will Added DMEM growing medium until the top of the scaffold material decalcifies with it he bone matrix is only just submerged, subsequent cultivation at 37°C and 5% CO». In 24 hours, an induction cultivation for osteogenic differentiation is carried out in vitro for 12-14 days. The osteogenic induction medium is: DMEM(H) + 10% FBS + 10 mmol/L B-glycero sodium phosphate + 0.1 µmol/L dexamethasone + 50 mg/L VitC. The scanning electron microscopic observations can show that the cells are successfully implanted onto the composite scaffold of hydroxyapatite and collagen. Subsequent freezing at -70°C to -80°C for 48-72 hours, freeze-drying for 24-30 hours and cryopreservation for 3 months to significantly reduce immunogenicity, thus becoming matrix-dependent tissue-engineered bone with osteoclast precursors and mesenchymal stem cells formed as germ cells.
    Exemplary embodiment 2 Detection of the biocompatibility of a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells
    1. Detection of the mixed lymphocyte culture reaction Mixed lymphocyte reaction (MLR) Obtaining BALB/c spleen lymphocytes from mice, pretreating for 3 hours with 50 g/L mitomycin C and resuspending in RPMI 1640 culture substrate as stimulator cells. collecting C57BL/6 spleen lymphocytes and
    Incubate with 0.5 µM CFSE at 37°C for 15 minutes to label them as reactive cells. The two are implanted at a 1:1 ratio in a 96-well plate for co-culture, 5x10° cells/well, resuspend in RPMI 1640 culture medium, then the following is added to the system: (1) BALB only /c monocytes are used as a negative control group; 2) the positive control group of mouse BALB/c spleen lymphocytes is treated with phytohemagglutinin (PHA); (3) a tissue engineered bone with MSC as gametes (using MSC as gametes implanted on the scaffold and then osteogenically induced in 96 pores for 14 days, without lyophilization); (4) a tissue engineered bone formed with the osteoclast precursors and the mesenchymal stem cells as germ cells (using POC and MSC as germ cells implanted on the scaffold material and then osteogenically induced in 96 pores for 14 days without freeze-drying); (5) a matrix-dependent tissue-engineered bone with only MSC as germ cells (freeze-dried overnight); (6) a matrix-dependent tissue-engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells (freeze-dried overnight). After culturing for 5 days, CCK-8 reagent is added. In 4 hours the proliferative ability of mononuclear cells of the peripheral blood is measured with a microplate reader at a wavelength of 450 nm. Figure 2 shows a detection result of a mixed lymphocyte culture reaction (MLR), as shown in Figure 2, the proliferation rates of the 6 groups are 2.23+0.27, 0.96+0.13, 0.89+0.11, 1, respectively .53+0.21, 0.41+0.06 and 0.52+0.07. Statistics show that a freeze-dried matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells has a significant difference in the stimulation index of peripheral blood mononuclear cells compared to the non-freeze-dried cell group (P<0.05), but no significant difference compared to matrix-dependent tissue-engineered bone with only MSC as germ cells. This indicates that the tissue engineered bone constructed by the present invention has low immunogenicity.
    2. MHC-I and MHC-II fluorescence detection
    In this experiment, the expression of class I and class II major histocompability complex (MHC) molecules of a matrix-dependent tissue engineered bone with osteoclast progenitors and mesenchymal stem cells as germ cells is explored. The expression of MHCI and MHCI was detected by immunofluorescence histochemistry technique. Figure 3 shows the expression results of MHC-I and MHC-II. The results show that a freeze-dried matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells shows a significant difference in MHC I and MHC II expression compared to the non-freeze-dried cell group (P<0.05), but none significant difference compared to matrix-dependent tissue-engineered bone with only MSC as germ cells. This indicates that the tissue engineered bone constructed by the present invention has low immunogenicity.
    Embodiment 3 Detection of in vitro migration, repair and osteogenic differentiation of matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells
    1. In vitro migration experiment: a 24-well plate is used for the cell migration experiment. Bone marrow MSCs are implanted in the upper chamber of the Transwell culture system at 10° cells/mL from 500 µL and the lower chamber is matrix-dependent tissue engineered bone constructed with the present invention with osteoclast precursors and mesenchymal stem cells as germ cells (MSC+ POC group), and a matrix-dependent tissue engineered bone with only MSC as germ cells is used as a control (MSC group). In 24 hours, the cells that migrated to the bottom of the filter membrane are fixed with 4% paraformaldehyde and stained with DAPI. The results are shown in Figure 4, the number of cell migrations in the MSC+POC group is 144.37+12.83, the number in the MSC group is 76.42+8.54, where the number in the MSC+POC group significantly higher than that in the MSC group (P<0.01).
    2. In vitro scratch repair experiment: seeding the endothelial cells (10° cells/pore) on a 6-well plate, and further incubating at 37°C, when the cells reach 90% growth density, the surface of the cells is mitigated a 200 µl pipette tip and wiped, final washing of cells with PBS to remove debris. Using, respectively, a matrix-dependent tissue-engineered bone constructed with the present invention with the osteoclast precursors and the mesenchymal stem cells as germ cells (MSC+POC group) and a matrix-dependent tissue-engineered bone with only MSC as germ cells (MSC group) that Both protein extracts and the culture medium containing 2% serum are mixed in a 1:1 ratio to continue the culture. Observe the changes in the scratched area, observing with a microscope at t=0 and 12 hours and measuring the size of the scratched area with Image J. Scratch Healing Rate (%) = (A0-A12)/A0 x 100%, where AO for the initial scratched area (t=0 hour) and A12 for the final scratched area at t=12 hours. Figure 5 shows the results of an MSC in vitro scratch repair experiment and in vitro osteogen differentiation experiment by a protein extract from a matrix-dependent tissue engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells. As shown in Figure 5, the proportion of Tissue repair in the MSC+POC group about 92% and in the MSC group about 78%. The MSC+POC group is significantly better than the MSC group (P<0.01).
    3. In vitro osteogenic differentiation experiment: take second generation bone marrow MSCs, when they reach 80%-90% growth density, they are digested with pancreatin for 5 minutes, centrifuge for 3 minutes at 1000 rpm, prepare the cell suspensions with F12 culture medium, seeding into a 24-well plate at 2x10°/pore, respectively using a matrix-dependent tissue engineered bone constructed with the present invention with the osteoclast precursors and the mesenchymal stem cells as germ cells (MSC+POC group ) and a matrix-dependent tissue-engineered bone with only MSC as germ cells (MSC group), the two protein extracts and the culture medium containing 10% serum are mixed in the ratio of 1:1 to continue the culture, in 21 days Alizarin red staining and ALP staining are performed, and the formation of calcium nodules is observed with an inverted microscope and roughly photographed. The results are shown in Figure 6. Alizarin red staining is used to assess the osteogenic differentiation ability of two types of matrix-dependent tissue-engineered bone. The MSC group mineralization ratio is approximately 40% and the MSC+POC group mineralization ratio is approximately 48%, with obvious differences. ALP staining is used to assess the osteogenic differentiation ability of two varieties of matrix-dependent tissue engineered bone. The MSC group mineralization ratio is approximately 32% and the MSC+POC group mineralization ratio is approximately 39%, with obvious differences.
    Working Example 4 Using matrix-dependent tissue engineering bone with osteoclast progenitors and mesenchymal stem cells as germ cells to repair bone defects Animal model preparation and experimental grouping: Taking healthy SD rats 2 months old with a body weight of (200+20)g. After anesthesia, the bilateral femoral condyles are exposed under aseptic conditions and a 3 mm diameter bone defect is created with a dental drill. The rats are each given the following 3 groups of bone defect repairing implants: (1) dummy control group: only the defect is subjected to the debridement treatment; (2) control group: a matrix-dependent tissue engineered bone with only MSC as germ cells; (3) Experimental group: a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells according to the present invention. Repeatedly adding hydrogen peroxide, iodophor and physiological saline to wash and sew the defect in layers, and the test animals are caged after waking up and injected with penicillin-streptomycin to prevent infection. Two months after the operation, micro-CT is performed, in accordance with the bone volume fraction (BV/TV), bone trabecular thickness (Tb.Th), number of bone trabeculae (Tb.N) and trabecular bone separation (Tb.Sp), the repair is performed assessed by bone defects. To the histological
    Upon observation, Masson's stain is performed to assess new bone formation at the defect site. Figure 7 shows the results of micro-CT examination 2 months after the implantation, the results show that 2 months after the operation defect repair is essentially complete in the experimental group and in the control group there is some new bone formation but not complete bone structure is seen at the defect site, with almost no new bone formation in the sham control group. In terms of bone volume fraction (BV/TV) and bone trabecular count (Tb.N), the experimental group is significantly better than the other two groups. Figure 8 shows the results of Masson's stain 2 months after the implantation, the Masson's stain of the experimental group shows a large amount of new bone tissue 2 months after the operation, the trabecular bone structure can be seen, and relatively many osteoblasts and chondrocytes can be partially seen will. In the control group, there is a small amount of lamellar bone trabecular structure, a small amount of osteoblasts and chondrocytes. In the blind control group, the fibrous tissue structure is more common and no obvious new bone is seen.
    Finally, it is pointed out that the above preferred embodiments are used to explain the technical solution of the present invention instead of limiting it, although in connection with the above preferred embodiments the present invention is explained in more detail for those skilled in the art to understand that various changes in form and details can be made without departing from the scope defined by the claims of the present invention.
    权利要求:
    Claims (8)
    [1]
    1. Construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells, characterized in that the mesenchymal stem cells and the osteoclast precursors are implanted at a ratio of 10: 1 on a porous skeleton material to form a tissue engineering complex to construct, then adding osteogenic induction culture medium in vitro, further culturing for 12-14 days, freezing for 48-72 hours at -70°C to -80°C, freeze-drying for 24-30 hours to generate matrix-dependent tissue to obtain engineering bones.
    [2]
    2. Construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells according to claim 1, characterized in that the mesenchymal stem cells are mesenchymal bone marrow stem cells, mesenchymal stem cells of peripheral blood, mesenchymal stem cells of umbilical cord blood, mesenchymal fat stem cells or mesenchymal stem cells of umbilical cord are.
    [3]
    3. Construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells according to claim 2, characterized in that the mesenchymal stem cells are mesenchymal stem cells of the umbilical cord.
    [4]
    4. Construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells according to claim 1, characterized in that the porous skeleton material is a decellularized bone matrix or a decalcified bone matrix.
    [5]
    5. Construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as
    Germ cells according to claim 4, characterized in that the porous skeletal material is a decalcified bone matrix.
    [6]
    6. Construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells according to any one of claims 1 to 5, characterized in that a scaffold material with decalcified bone matrix is taken, soaking this for 48 hours in the DMEM culture substrate, setting the pH to 7.2; Take second generation mesenchymal stem cells in exponential growth phase, wash them with PBS and incubate for one hour using 0.1 wt% collagen enzyme type I solution, then add 0.25 wt% pancreatin and 0.02 wt% wt% EDTA and digest for 3-5 minutes, wash in DMEM culture medium containing 10 wt% fetal calf serum and resuspend, and mix with the osteoclast precursors at a ratio of 10:1 and effect resuspension, inoculate the cells onto the scaffold with decalcified bone matrix after the above treatment so that the cell suspension only infiltrates the scaffold and does not overflow the scaffold; and wherein in 3 hours the underside of the scaffold material with decalcified bone matrix is turned over to the top side under aseptic operating conditions and the cell suspension is inoculated by the above method; and wherein the cell scaffold has a regular size of 0.5cm x 0.5cm x 0.3cm and the total number of cells implanted is about 10°, and in 3 hours the DMEM culture substrate is added until the top of the framework material with decalcified bone matrix is only just submerged, subsequent cultivation at 37°C and 5% CO»; Performing induction cultivation for osteogenic differentiation in 24 hours in vitro for 12-14 days, freezing at -70°C to -80°C for 48-72 hours, freeze-drying for 24-30 hours and cryopreservation for 3 months to significantly increase the immunogenicity to reduce, thereby forming a matrix-dependent tissue-engineered bone with osteoclast precursors and mesenchymal stem cells as germ cells.
    [7]
    7. Construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells according to claim 6, characterized in that the osteoclast precursors are derived from bone marrow monocytes for 24 hours by 100 ng / ml RANKL and 50 ng / ml M-CSF be differentiated in the osteoclast direction.
    [8]
    8. Matrix-dependent tissue engineered bone having osteoclast progenitors and mesenchymal stem cells as germ cells constructed by the method of any one of claims 1 to 7.
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    CN109224130B|2018-10-09|2021-04-20|中国人民解放军陆军军医大学|Application of long-chain non-coding RNA lnc-HCAR in preparation of bone repair system, bone repair system and preparation method|
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    CN202010085219.1A|CN111214707A|2020-02-10|2020-02-10|Matrix-dependent tissue engineering bone with osteoclast precursor and mesenchymal stem cell as seed cells and construction method thereof|
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