BE1028040A1 - Matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells and methods of construction therefor - Google Patents

Abstract

The present invention discloses a matrix-dependent tissue engineering bone having osteoclast precursors and mesenchymal stem cells as germ cells and a construction method therefor, wherein the mesenchymal stem cells and the osteoclast precursors are implanted at a ratio of 10: 1 on a porous skeleton material to produce a tissue engineering Construct a complex, then an osteogenic induction culture medium is added in vitro, further cultivation 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 Get engineering bones. The in vivo research shows that the present invention can promote new bone formation and successfully repair the bone defects.

Description

Matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells and methods of construction therefor

TECHNICAL FIELD The present invention relates to the technical field of tissue engineering and a matrix-dependent tissue engineering bone having osteoclast precursors and mesenchymal stem cells as germ cells and a construction method therefor.

BACKGROUND ART The treatment of large bone defects has always been one of the difficult problems in clinical treatment. Autogenous bone is the "gold standard" for bone transplantation, but this is part of an invasive repair mode and cannot meet the enormous clinical need for bone repair materials. Subsequently, a variety of bone substitute materials will be developed such. Xenogeneic bone, allogeneic decalcified bone, and artificial bone substitute, etc. However, the presence of xenogeneic bone has deficiencies in that it has poor bone graft survival and is prone to immune rejection and the spread of disease, which are difficult to meet clinical needs can. The greatest shortcoming of allogeneic decalcified bones is that disease spread is easy, large bone is difficult to form in the recipient after transplant, 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 highly active bone repair materials for children and young patients, and has achieved good clinical results and provided good bone repair materials for patients with autogenous bone deficiency. The advantage is that only 10 mL of the patient's bone marrow should be removed, a large number of germ cells are multiplied in vitro and then inoculated on the scaffold material in order to construct the tissue engineering bone. After cultivation to maturity, a large amount of highly active bone repair materials can be obtained. The method is suitable for children and patients with a strong ability to multiply stem cells in autogenous bone marrow, and its use in the elderly, in particular in patients with underlying diseases, is restricted. Limitations include: 1) long construction time, requiring nearly 3 weeks of in vitro amplification and cultivation; 2) high technical requirements, which requires specialized operating rooms with 100 levels and qualified professionals; 3) high clinical cost, each case requires high cultivation costs on average. The core of the osteogenic activity of this conventional tissue engineering bone resides in the germ cells, but there are problems with storing and transporting the tissue engineering bone and maintaining germ cell activity. In addition, there are many technical links and quality control is difficult to achieve. It is only used in individual hospitals and individual groups. The large-scale clinical application and the realization of industrialization will be restricted.

The use of abundant proteins and factors in the extracellular matrix to repair tissue damage is a current focus of research. At an early stage, a bone matrix material containing a large amount of proteins secreted by mesenchymal stem cells of the umbilical cord and a manufacturing process (patent ZL 2013 1 0449152.5) were established, and on this basis a design concept and system for a matrix-dependent one were established Tissue engineering bone "Matrix Based Tissue Engineering Bone, M-TEB)" developed. The basic construction strategy is as follows: the mesenchymal stem cells (MSC) are connected to bone framework materials, cultivation for a total of 14 days, freezing of the cell-framework complex for 48 hours at -80 ° C and freeze-drying for 24 hours MSC to form ECM-TEB of the germ cells. With this construction technology, the cytokines and remain

Matrix proteins, which wrap the cells in an autocrine manner on the scaffold material, are retained even though the cell activity is removed. After in vivo transplantation, the cytokines wrapped in the matrix are slowly released at the injury site under the action of enzymes and take part in bone regeneration and reconstruction. Because of this, the biologically active protein charged and released by the decellularized M-TEB fulfills the requirements of the physiological environment.

The 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 the bone tissue. Therefore, if different cell types can be co-cultivated in the construction of ECM-TEB, the microenvironment of bone formation in vivo will be simulated more realistically. The two ultimate functional cells required to maintain bone homeostasis are osteoblasts and osteoclasts. The osteoblasts (osteoblast, OB) come 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 the cleavage of the monocyte / macrophage cell line in hematopoietic stem cells and complete the bone absorption and breakdown through local acid secretion and the release of the enzymes that break down the bone matrix. The two cell types work closely together through precise regulation to complete bone tissue reconstruction and maintain bone homoostasis. The studies have shown that PDGF-BB, secreted by osteoclast precursors (pre-osteoclasts, POCs) can promote the coupling of bone formation and vascularization, suggesting that osteoclast-related cells play a key role in bone repair and vascularization. This provides a theoretical basis for the introduction of osteoclast precursors as "composite" germ cells in the present invention, while MSCSs are used as germ cells for osteogenic differentiation.

There is currently no report of osteoclast precursors as one of the germ cells for the construction of matrix-dependent tissue engineering bone and for the treatment of bone defects.

SUMMARY OF THE PRESENT INVENTION In view of the foregoing, the object of the present invention is to establish a novel tissue engineering bone construction with current use of a large tissue engineering bone for repairing large bone defects in order to overcome the bottleneck problem with the dependence on autogenous cells and living Cells using conventional tissue engineering 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 method of construction therefor. The present invention employs the following technical solution:

1. A construction method for a matrix-dependent tissue engineering bone having 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 skeleton 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.

The mesenchymal stem cells are preferably mesenchymal bone marrow stem cells, mesenchymal stem cells of the peripheral blood, mesenchymal stem cells of the umbilical cord blood, mesenchymal fat stem cells or mesenchymal stem cells of the umbilical cord.

The mesenchymal stem cells are preferably mesenchymal stem cells of the umbilical cord.

The porous bone framework material is preferably a decellularized bone matrix or a decalcified bone matrix. The porous bone framework material is preferably a decalcified bone matrix.

5 Preferably, a framework material with decalcified bone matrix is taken, this is soaked for 48 hours in the DMEM culture substrate, the pH value is adjusted to 7.2; Take the second generation of mesenchymal stem cells in the exponential growth phase, wash them with PBS and incubate for one hour using 0.1% by weight collagen enzyme solution of type L, then adding 0.25% by weight pancreatin and 0.02% by weight % EDTA and digest 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 performing resuspension; Inoculating the cells onto the framework material with decalcified bone matrix after the above-mentioned treatment, so that the cell suspension only infiltrates the framework material and does not overflow the framework material.In 3 hours, under aseptic operating conditions, the underside of the framework material with decalcified bone matrix is turned upside down, and the cell suspension is inoculated using the procedure outlined above; the cell framework has a regular size of 0.5 cm x 0.5 cm x 0.3 cm and the total number of implanted cells is about 10 °, the DMEM culture substrate is added in 3 hours until the top of the framework material with decalcified bone matrix is only just immersed, subsequent cultivation at 37 ° C and 5% CO ». Perform 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 make immunogenicity significant to reduce, in this way a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells is formed.

The osteoclast precursors are preferably derived from bone marrow monocytes, which are differentiated for 24 hours by 100 ng / mL RANKL and 50 ng / mL M-CSF in the direction of the osteoclast.

2. Using the above method, a 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 extends the construction method of the matrix-dependent tissue engineering bone but also a more realistic one Construct osteogenic microenvironment in vivo. The results of in vivo studies show that the matrix-dependent tissue engineering bone constructed by the method of the present invention with 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 In connection with the following figures, the present invention is explained in more detail so that the aim, the technical solution and the advantages of the present invention become clearer.

FIG. 1 shows a schematic diagram of a strategy of a construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells.

FIG. 2 shows a diagram of the detection result of a mixed lymphocyte culture reaction (MLR).

FIG. 3 shows a diagram of the fluorescence detection results of MHC-I and MHC-II of a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells.

FIG. 4 shows a diagram of the results of an in vitro migration experiment of endothelial cells through a protein extract from a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells.

FIG. 5 shows a diagram of the results of an MSC in vitro scratch repair experiment and in vitro osteogen differentiation experiment using a protein extract from a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells.

FIG. 6 shows a diagram of the results of an MSC in vitro osteogen differentiation experiment using a protein extract from a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells.

FIG. 7 shows a diagram of the micro-CT examination results of a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells in 2 months after planting for the repair of femoral defects in rats.

FIG. 8 shows a diagram of the results of the Masson staining of a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells in 2 months after planting for the 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 in specific conditions are not given, the experiments are usually carried out according to conventional conditions or according to the conditions recommended by the reagent manufacturer.

Embodiment 1: Construction of a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells

1. Culturing Umbilical Cord MSC Take a healthy fetal umbilical cord, cut off the arteries and veins, and dissect; Culturing using a tissue block, digesting the umbilical cord with type I collagenase; Washing the tissue block with a phosphate buffer solution, transferring the tissue block to a culture flask, and adding the culture substrate to carry out a culture adhering to the wall; Cultivate in an incubator with a constant temperature of 37 ° C and 5% CO », change the substrate every 3 days, after changing the substrate, the 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 on at a ratio of 1: 2. When the above-mentioned cultured bone marrow MSCs are fused to about 80%, 2.5 g / L trypsin and 0.2 g / L EDTA are mixed at a ratio of 1: 1 and digested. The cells are placed in a subculture flask (T25) with a cell density of 8.0 x 10% / cm? inoculated for propagation and cultivation. Cell growth and morphological properties are observed under an inverted microscope. The results show that the second generation cells grew well and had a uniform morphology. Most cells are mature mesenchymal stem cells in wide large polygons or flat shapes.

2. Culturing of osteoclast precursors The primary bone marrow macrophages (BMMs) derived from bone marrow are used for induction culture. BMMs are inoculated evenly into a cell culture plate (5000 cells / pore, 96-pore 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 obtained, which is assessed and confirmed by the TRAP staining, the CFA staining, 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 engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells The construction strategy is as shown in FIG. Place in a 6-pore plate, soak this for 48 hours in the DMEM culture substrate, adjust the pH to about 7.2; Take the second generation of MSCs in the exponential

Growth phase, washing this with PBS for two times and then incubating for one hour using 0.1% collagen enzyme type I (Col D, then adding 0.25% pancreatin and 0.02% EDTA and digesting for 3-5 Minutes, washing in DMEM culture substrate and resuspending, and mixing with the osteoclast precursors at a ratio of 10: 1 and performing a resuspension.Inoculation of the cells onto the framework material with decalcified bone matrix after the above treatment so that the cell suspension only infiltrates the framework material and the framework material does not overflow. In 3 hours, the underside of the framework material with decalcified bone matrix is turned over to the top under aseptic operating conditions, and the cell suspension is inoculated using the method described above, the total number of implanted cells is about 10 °. In 3 hours this will be DMEM growing medium added until the upper side of the framework material decalcified with the bone matrix is only just immersed, subsequent cultivation at 37 ° C and 5% CO ». Induction cultivation for osteogenic differentiation in vitro for 12-14 days is carried out in 24 hours. The osteogenic induction medium is: DMEM (H) + 10% FBS + 10mmol / L B-glycerone sodium phosphate + 0.1 µmol / L dexamethasone + 50 mg / L VitC. The scanning electron microscopic observations can show that the cells are successfully implanted on the composite framework material made 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 in order to significantly reduce the immunogenicity, in this way a matrix-dependent tissue engineering bone with osteoclast precursors becomes and mesenchymal stem cells are formed as germ cells.

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. Collect C57BL / 6 spleen lymphocytes and

Incubate with 0.5 µM CFSE at 37 ° C for 15 minutes to mark them as reactive cells. The two are implanted in a 96-pore plate for co-culture at a ratio of 1: 1, 5x10 ° cells / pore, resuspended in RPMI 1640 culture substrate, 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 BALB / c spleen lymphocytes from mice is treated with phytohemagglutinin (PHA); (3) a tissue engineering bone with MSC as germ cells (using MSC as germ cells, which are implanted on the scaffold and then osteogenically induced in 96 pores for 14 days without lyophilization); (4) a tissue engineering bone formed with the osteoclast precursors and the mesenchymal stem cells as germ cells (using POC and MSC as germ cells, which are implanted on the scaffold and then osteogenically induced in 96 pores for 14 days without Freeze-drying); (5) a matrix-dependent tissue engineering bone with only MSC as germ cells (freeze-dried overnight); (6) a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells (freeze-dried overnight). After culturing for 5 days, CCK-8 reagent is added. The ability of mononuclear cells of the peripheral blood to multiply is measured in 4 hours with a microplate reader at a wavelength of 450 nm. FIG. 2 shows a detection result of a mixed lymphocyte culture reaction (MLR), as shown in FIG. 2, the multiplication 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 freeze-dried matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells shows a significant difference in the stimulation index of mononuclear cells of the peripheral blood compared to the non-freeze-dried cell group (P <0.05), but no significant difference compared to matrix-dependent tissue engineering bone with only MSC as germ cells. This indicates that the tissue engineering bone constructed by the present invention has low immunogenicity.

2. MHC-I and MHC-II fluorescence detection

This experiment investigates the expression of class I and class II molecules of the major histocompatibility complex (MHC) of a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells. The expression of MHCI and MHCI was detected by the immunofluorescence histochemistry technique. FIG. 3 shows the expression results of MHC-I and MHC-II. The results show that freeze-dried matrix-dependent tissue engineering 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 engineering bone with only MSC as germ cells. This indicates that the tissue engineering bone constructed by the present invention has low immunogenicity.

Embodiment 3 Recognition of the in vitro migration, repair and osteogenic differentiation of a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells

1. In vitro migration experiment: a 24-pore plate is used for the cell migration experiment. Bone marrow MSCs are implanted in the upper chamber of the Transwell culture system with 10 ° cells / mL of 500 uL, and the lower chamber is a matrix-dependent tissue engineering bone constructed with the present invention with osteoclast precursors and mesenchymal stem cells as germ cells (MSC + POC group), and a matrix-dependent tissue engineering 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 FIG. 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, the number in the MSC + POC group significantly higher than that in the MSC group (P <0.01).

2. In vitro scratch repair experiment: Inoculation of the endothelial cells (10 ° cells / pore) on a 6-pore plate, and further incubation at 37 ° C, when the cells reach a growth density of 90%, the surface of the cells becomes with scraped and wiped with a 200 µl pipette tip, final washing of cells with PBS to remove splinters. Each use of a matrix-dependent tissue engineering 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 engineering bone only with MSC as germ cells (MSC group), which Both protein extracts and the culture medium, which contains 2% serum, are mixed in a ratio of 1: 1 to continue the culture. Observing 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 stands for the final scratched area at t = 12 hours. FIG. 5 shows the results of an MSC in vitro scratch repair experiment and in vitro osteogen differentiation experiment using a protein extract from a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells. As shown in FIG 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% to 90% growth density, digested with pancreatin for 5 minutes, centrifuge for 3 minutes at 1000 rpm, prepare of the cell suspensions with F12 culture substrate, inoculation into a 24-pore plate at 2 × 10 ° / pore, in each case using a matrix-dependent tissue engineering 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 engineering bone with only MSC as germ cells (MSC group), the two protein extracts and the culture substrate, which contains 10% serum, are mixed in a ratio of 1: 1 to continue the culture in 21 days Alizarin red staining and ALP staining are carried out, and the formation of calcium nodules is observed with an inverted microscope and roughly photographed. The results are shown in FIG. Alizarin red staining is used to assess the osteogenic differentiation ability of two types of matrix-dependent tissue engineering bone. The mineralization ratio of the MSC group is about 40% and the mineralization ratio of the MSC + POC group is about 48%, there are obvious differences. The ALP staining is used to assess the osteogenic differentiation ability of two types of matrix-dependent tissue engineering bone. The mineralization ratio of the MSC group is about 32% and the mineralization ratio of the MSC + POC group is about 39%, there are obvious differences.

Embodiment 4 Use of a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells for the repair of bone defects. Animal model preparation and experimental grouping: Take healthy SD rats, which are 2 months old, with a body weight of (200 + 20) g. After anesthesia, the bilateral femoral condyles are exposed under aseptic conditions and a bone defect 3 mm in diameter is made with a dental drill. The rats each receive the following 3 groups of implants for the repair of bone defects: (1) Blind control group: the debridement treatment is only carried out for the defect; (2) control group: a matrix-dependent tissue engineering bone with only MSC as germ cells; (3) Experiment group: a matrix-dependent tissue engineering bone having osteoclast precursors and mesenchymal stem cells as germ cells according to the present invention. Repeated addition of hydrogen peroxide, iodophor, and saline to wash and sew the defect in layers, and after awakening the test animals are caged 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), the thickness of the trabecular bone (Tb.Th), the number of trabecular bones (Tb.N) and the trabecular bone separation (Tb.Sp), the repair is carried out assessed by bone defects. For histological

Observation, a Masson stain is performed to assess the formation of new bone at the defect site. FIG. 7 shows the results of the micro-CT examination 2 months after the implantation, the results show that 2 months after the operation, the defect repair in the experiment group is essentially complete and in the control group there is somewhat new bone formation, but not a complete bone structure is seen at the defect site with almost no new bone formation in the blind control group. With regard to the bone volume fraction (BV / TV) and the number of bone trabeculae (Tb.N), the experimental group is significantly better than the other two groups. Figure 8 shows the results of Masson staining 2 months after the implantation, the Masson staining 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 trabecular structure, a small amount of osteoblasts and chondrocytes. In the blind control group, the fibrous tissue structure is more common and no apparent new bone is seen.

Finally, it is pointed out that the above preferred exemplary embodiments are used to explain the technical solution of the present invention instead of restricting it, although the present invention is explained in more detail in connection with the above preferred exemplary embodiments, should be understood by the person skilled in the art that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims (8)

Claims 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 with a ratio of 10: 1 on a porous bone framework material to form a tissue engineering complex to construct, in which case an osteogenic induction culture medium is added in vitro, further cultivation for 12-14 days, freezing for 48-72 hours at -70 ° C to -80 ° C, freeze-drying for 24-30 hours in order to obtain a matrix-dependent tissue Get engineering bones. 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 the peripheral blood, mesenchymal stem cells of the umbilical cord blood, mesenchymal stem cells of the umbilical cord blood or mesenchymal fatty stem cells are. 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. 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 bone framework material is a decellularized bone matrix or a decalcified bone matrix. 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 bone framework material is a decalcified bone matrix. 6. Construction method for a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells according to one of claims 1 to 5, characterized in that a framework material with decalcified bone matrix is taken, soaking this for 48 hours in the DMEM culture substrate, setting the pH to 7.2; Take the second generation of mesenchymal stem cells in the exponential growth phase, wash them with PBS and incubate for one hour using 0.1 wt% collagen enzyme solution type I, then adding 0.25 wt% pancreatin and 0.02 % By weight EDTA and digesting for 3-5 minutes, washing in DMEM culture substrate containing 10% by weight fetal calf serum and resuspending, and mixing with the osteoclast precursors at a ratio of 10: 1 and performing resuspension, inoculation of the cells on the framework material with decalcified bone matrix after the above-mentioned treatment, so that the cell suspension only infiltrates the framework material and does not overflow the framework material; and wherein, in 3 hours, the underside of the framework material with decalcified bone matrix is turned over to the top under aseptic operating conditions, and the cell suspension is inoculated using the method described above; and wherein the cell scaffold has a regular size of 0.5 cm x 0.5 cm x 0.3 cm and the total number of implanted cells is about 10 °, and wherein the DMEM culture substrate is added in 3 hours until the top of the Framework material with decalcified bone matrix is only just immersed, subsequent cultivation at 37 ° C and 5% CO »; Perform 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 make immunogenicity significant to reduce, whereby a matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells is formed. 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 originate from bone marrow monocytes which for 24 hours by 100 ng / mL RANKL and 50 ng / mL M-CSF differentiated in the direction of the osteoclast. 8. Matrix-dependent tissue engineering bone with osteoclast precursors and mesenchymal stem cells as germ cells, which is constructed using the method according to any one of claims 1 to 7.