CN106581066B - Uses of tissue engineered bone - Google Patents

Abstract

The invention discloses the use of tissue engineering bone in preparing medicine for treating bone injury diseases, and the tissue engineering bone includes mesenchymal stem cells and pre-induced cells. The tissue engineered bone of the present invention has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, and can provide the necessary cells for tissue repair for the damaged part of the bone, so that the damaged part of the bone can be repaired and healed quickly and effectively. And easy to obtain, safe and reliable.

Description

Uses of tissue engineered bone

technical field

The present invention relates to the field of medicine. In particular, the present invention relates to tissue engineered bone uses.

Background technique

Bone injury often occurs when the fracture fragment penetrates the limb during trauma or when the debridement of an open fracture is removed, and it mainly includes two major parts: bone defect and defect repair. Bone injury is a common clinical disease and one of the difficult problems in orthopedic treatment.

However, current drugs for bone-injuring diseases remain to be developed.

SUMMARY OF THE INVENTION

The present invention aims to solve one of the technical problems existing in the prior art at least to a certain extent. To this end, the present invention proposes the use of tissue engineered bone in the preparation of medicines.

It should be noted that the present invention is accomplished based on the following findings of the inventors:

At present, the commonly used clinical treatment methods mainly include autologous bone transplantation, allogeneic bone transplantation, xenografted bone transplantation and artificial bone transplantation, among which autologous bone transplantation is considered as the gold standard for repairing bone defects. However, since autologous bone transplantation is a kind of digging the east wall to make up the west wall, it not only provides a very limited amount of bone, but also may cause problems such as trauma, deformity, pain and infection in the donor site, and more importantly, cause new bone defects; The source of bone is limited, and there is a risk of rejection and infection of infectious diseases such as viral hepatitis and AIDS; simple artificial bone transplantation has very low osteogenic efficiency due to the lack of cells necessary for tissue repair. Moreover, for patients with delayed bone union and nonunion, the above methods are often ineffective, and nonunion and delayed bone union still exist after surgery.

In the research on the treatment methods of bone injury diseases, the inventors found that based on the characteristics of stem cells with high self-renewal ability, high proliferation and multi-directional differentiation potential, implantability and reconstruction ability, they can be cultured in vitro and expanded, It can effectively treat bone injury diseases when it is injected into the site of bone injury.

The inventors conducted in-depth research and found that by pre-inducing mesenchymal stem cells with the ability to differentiate into osteocytes, pre-induced cells with strong ability to differentiate into osteocytes can be obtained. Further, the inventors found that when mesenchymal stem cells and pre-induced cells act together on the bone injury site, the repair and healing efficiency of the bone injury site is significantly higher than that of the pre-induced cells alone. Thus, the tissue engineered bone containing mesenchymal stem cells and pre-induced cells has a strong ability to proliferate and differentiate into osteocytes, and can repair and heal the damaged part of the bone quickly and effectively.

To this end, the present invention proposes the use of tissue engineered bone in the preparation of medicines. According to an embodiment of the present invention, the medicine is used for the treatment of bone injury diseases, and the tissue engineered bone comprises: mesenchymal stem cells and pre-induced cells.

The inventors found that mesenchymal stem cells have the ability to differentiate into bone cells, but the direction of differentiation is uncontrollable. Furthermore, by pre-inducing the cells, pre-induced cells having a strong ability to differentiate into osteocytes are obtained, whereby the pre-induced cells can be effectively differentiated into osteocytes, and the yield of osteocytes is high. The inventors unexpectedly found that when pre-induced cells and mesenchymal stem cells are used together, the yield of bone cells is higher than that of pre-induced cells alone. After in-depth research, it was found that mesenchymal stem cells can not only differentiate into bone cells and increase the number of bone cells, but also produce some promoting factors during the differentiation process of mesenchymal stem cells, which can promote the proliferation and differentiation of pre-induced cells, and improve the overall Osteocyte yield. Therefore, the tissue-engineered bone according to the embodiment of the present invention has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, and can provide the necessary cells for tissue repair for the damaged part of the bone, so that the damaged part of the bone can be rapidly and effectively repaired. Effectively repairs and heals, and is easily accessible, safe and reliable.

According to an embodiment of the present invention, the use of the above-mentioned tissue engineered bone in the preparation of medicine may also have the following additional technical features:

According to an embodiment of the present invention, the administration mode of the drug is injection.

According to an embodiment of the present invention, the content of the mesenchymal stem cells is 1×10 ⁷ to 1×10 ⁸ cells/mL, and the content of the pre-induced cells is 1×10 ⁷ to 1×10 ⁸ cells/mL. Therefore, tissue-engineered bone has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, which can provide the necessary cells for tissue repair for the bone injury site, so that the bone injury site can be repaired and healed quickly and effectively. And easy to obtain, safe and reliable.

According to an embodiment of the present invention, the number ratio of the mesenchymal stem cells to the pre-induced cells is 1:1-4, preferably 1:2. The inventors obtained the above proportional relationship through a large number of experiments. Therefore, the tissue engineered bone has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, which can provide the necessary cells for tissue repair for the damaged part of the bone, so that the bone can be repaired. The injury site is repaired and healed quickly and effectively, and it is easy to obtain, safe and reliable. If there are too many mesenchymal stem cells, the yield of differentiated osteocytes will be low; if there are too few mesenchymal stem cells, the yield of differentiated osteocytes will also be low.

According to an embodiment of the present invention, the mesenchymal stem cells are bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, amniotic fluid stem cells or dental pulp stem cells, preferably umbilical cord mesenchymal stem cells. Umbilical cord mesenchymal stem cells are easy to obtain. Therefore, the tissue-engineered bone according to the embodiment of the present invention has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, and can provide the necessary cells for tissue repair for the damaged part of the bone, so that the damaged part of the bone can be rapidly and effectively repaired. Effectively repairs and heals. In addition, the tissue-engineered bone is in liquid state, which is easy to operate, easy to obtain, safe and reliable.

It can be understood by those skilled in the art that the umbilical cord mesenchymal stem cells are isolated, cultured and purified from umbilical cord-derived cells, and can be obtained commercially. Similarly, the sources of bone marrow mesenchymal stem cells, amniotic fluid stem cells or dental pulp stem cells will not be repeated here.

According to an embodiment of the present invention, the tissue engineered bone further comprises at least one of the following: 2×10 ¹⁰ /mL platelet-rich plasma; 1% w/v hydrogel; and 100 μM tris(2-dimethylaminoethyl) )amine. There are a variety of growth factors in platelet-rich plasma, and the proportion of each growth factor is in line with the normal proportion in the human body, so that there is the best synergy between the growth factors, and platelet-rich plasma contains a large amount of fibrin, which provides a good source for repairing cells. bracket. The addition of hydrogel can promote the adhesion of damaged cells and accelerate healing. Tris(2-dimethylaminoethyl)amine can accelerate the osteogenic differentiation of cells. Therefore, the tissue-engineered bone according to the embodiment of the present invention has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, and can provide the necessary cells for tissue repair for the damaged part of the bone, so that the damaged part of the bone can be rapidly and effectively repaired. Effectively repairs and heals. In addition, the tissue-engineered bone is in liquid state, which is easy to operate, easy to obtain, safe and reliable.

According to an embodiment of the present invention, the mesenchymal stem cells express the surface marker protein CD51. The inventors found that the mesenchymal stem cells that can express the surface marker protein CD51 have a strong ability to differentiate into bone cells.

According to an embodiment of the present invention, the pre-induced cells are obtained by the following methods: contacting the mesenchymal stem cells to be induced with an expansion medium to perform expansion culture, so as to obtain an expansion culture product; and The boosted culture product is contacted with the pre-induction medium, and the pre-induction culture is performed to obtain the pre-induced cells. Therefore, the tissue-engineered bone according to the embodiment of the present invention has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, and can provide the necessary cells for tissue repair for the damaged part of the bone, so that the damaged part of the bone can be rapidly and effectively repaired. Effectively repairs and heals. In addition, the tissue-engineered bone is in liquid state, which is easy to operate, easy to obtain, safe and reliable.

It should be noted that the source of the mesenchymal stem cells to be induced and the mesenchymal stem cells constituting the tissue engineered bone are not strictly limited, and they can be the same source, that is, the mesenchymal stem cells are divided into two parts, and one part is formed by pre-induction. Pre-induced cells, another part and pre-induced cells form tissue engineered bone; they can also be from different sources, such as belonging to different batches, different manufacturers, etc.

According to an embodiment of the present invention, contacting the expanded culture product with the pre-induction medium is to contact the expanded culture product at a rate of 3×10 ³ to 8×10 ³ /cm ² , preferably 5×10 ³ /cm 2 . The inoculum size of cm ² was inoculated in the pre-induction medium. The inventors obtained the above optimal inoculation amount through a large number of experiments, which can meet the needs of cell proliferation under this condition.

According to an embodiment of the present invention, the pre-induction culture time is 6-8 days, preferably 7 days. The inventors found that the cells required for induction are suitable for transplantation when the induction is cultured for 6 to 8 days. If the time is too long, the cells will be excessively differentiated and even die.

According to an embodiment of the present invention, the expansion medium comprises: α-MEM medium containing 10% by volume of fetal bovine serum. The inventors found that the use of a culture medium comprising α-MEM medium containing 10% by volume of fetal bovine serum can effectively make mesenchymal stem cells proliferate and facilitate subsequent pre-induction treatment.

It should be noted that the "α-MEM culture solution" is well known to those skilled in the art, and the obtaining method is not strictly limited, such as self-preparation or commercially available. According to the embodiment of the present invention, the α-MEM culture medium was purchased from Gibco, model number 12561-056.

According to an embodiment of the present invention, the pre-induction medium comprises: a basal medium, the basal medium comprises: DMEM medium containing 10% by volume fetal bovine serum; 50 μM ascorbic acid; 10 mM sodium β-glycerophosphate; and 0.1 mM Dexamethasone. The inventors obtained the above-mentioned optimal induction medium through a large number of experiments. In this medium, pre-induced cells can be effectively induced to form, and the induction efficiency is high.

For the convenience of understanding, the method for preparing the above-mentioned tissue engineered bone will be described in detail below:

According to an embodiment of the present invention, the method comprises: (1) pre-inducing mesenchymal stem cells to be induced to obtain pre-induced cells; and (2) mixing the pre-induced cells and mesenchymal stem cells, in order to obtain the tissue engineered bone.

The inventors found that mesenchymal stem cells have the ability to differentiate into bone cells, but the direction of differentiation is uncontrollable. Furthermore, by pre-inducing the cells, pre-induced cells having a strong ability to differentiate into osteocytes are obtained, whereby the pre-induced cells can be effectively differentiated into osteocytes, and the yield of osteocytes is high. The inventors unexpectedly found that when pre-induced cells and mesenchymal stem cells are used together, the yield of bone cells is higher than that of pre-induced cells alone. After in-depth research, it has been found that mesenchymal stem cells can not only differentiate into osteocytes and increase the number of osteocytes, but also produce some promoting factors during the differentiation process of mesenchymal stem cells to promote the proliferation and differentiation of pre-induced cells, thereby improving bone growth. cell yield. Therefore, the tissue engineered bone obtained by the method for preparing tissue engineered bone according to the embodiment of the present invention has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, which can provide the necessary tissue repair for the bone injury site. The cells can repair and heal bone damage quickly and effectively, and it is easy to obtain, safe and reliable.

According to an embodiment of the present invention, the number ratio of the mesenchymal stem cells to the pre-induced cells is 1:1-4, preferably 1:2. The inventors obtained the above proportional relationship through a large number of experiments, thus, the obtained tissue engineered bone has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, and can provide the necessary cells for tissue repair for the bone injury site. , so that the bone injury site can be repaired and healed quickly and effectively, and it is easy to obtain, safe and reliable. If there are too many mesenchymal stem cells, the yield of differentiated osteocytes will be low; if there are too few mesenchymal stem cells, the yield of differentiated osteocytes will also be low.

According to an embodiment of the present invention, the pre-induction treatment time is 6-8 days, preferably 7 days. The inventors found that the cells required for induction are suitable for transplantation when the induction is cultured for 6 to 8 days. If the time is too long, the cells will be excessively differentiated and even die.

According to an embodiment of the present invention, step (2) further comprises: adding at least one of platelet-rich plasma, hydrogel and tris(2-dimethylaminoethyl)amine, the mesenchymal stem cells and the pre-induced The cells are mixed, and the resulting mixed cells are resuspended in physiological saline to obtain the tissue engineered bone. There are a variety of growth factors in platelet-rich plasma, and the proportion of each growth factor is in line with the normal proportion in the human body, so that there is the best synergy between the growth factors, and platelet-rich plasma contains a large amount of fibrin, which provides a good source for repairing cells. bracket. The addition of hydrogel can promote the adhesion of damaged cells and accelerate healing. Tris(2-dimethylaminoethyl)amine can accelerate the osteogenic differentiation of cells. Therefore, the tissue engineered bone obtained according to the method of the embodiment of the present invention has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, and can provide the necessary cells for tissue repair for the bone injury site, so that the bone can be repaired. The injured area is repaired and healed quickly and effectively. In addition, the tissue-engineered bone is in liquid state, which is easy to operate, easy to obtain, safe and reliable.

According to an embodiment of the present invention, the pre-induction treatment includes: contacting the mesenchymal stem cells to be induced with an expansion medium to perform expansion culture, so as to obtain an expansion culture product; and combining the expansion culture product with a pre-expanded culture product The induction medium is contacted, and a pre-induction culture is performed to obtain the pre-induced cells. Therefore, the tissue engineered bone obtained according to the method of the embodiment of the present invention has a strong ability to differentiate into osteocytes, and the yield of osteocytes is high, and can provide the necessary cells for tissue repair for the bone injury site, so that the bone can be repaired. The injured area is repaired and healed quickly and effectively. In addition, the tissue-engineered bone is in liquid state, which is easy to operate, easy to obtain, safe and reliable.

According to an embodiment of the present invention, contacting the expanded culture product with the pre-induction medium is to contact the expanded culture product at a rate of 3×10 ³ to 8×10 ³ /cm ² , preferably 5×10 ³ /cm 2 . The inoculum size of cm ² was inoculated in the pre-induction medium. The inventors obtained the above optimal inoculation amount through a large number of experiments, which can meet the needs of cell proliferation under this condition.

According to an embodiment of the present invention, the expansion medium comprises: α-MEM medium containing 10% by volume of fetal bovine serum. The inventors found that the use of a culture medium comprising α-MEM medium containing 10% by volume of fetal bovine serum can effectively make mesenchymal stem cells proliferate and facilitate subsequent pre-induction treatment.

It should be noted that the "α-MEM culture solution" is well known to those skilled in the art, and the obtaining method is not strictly limited, such as self-preparation or commercially available. According to the embodiment of the present invention, the α-MEM culture medium was purchased from Gibco, model number 12561-056.

According to an embodiment of the present invention, the pre-induction medium comprises: a basal medium, the basal medium comprises: DMEM medium containing 10% by volume fetal bovine serum; 50 μM ascorbic acid; 10 mM β-glycerol phosphate; Semethasone. The inventors obtained the above-mentioned optimal induction medium through a large number of experiments. In this medium, pre-induced cells can be effectively induced to form, and the induction efficiency is high.

According to an embodiment of the present invention, the mesenchymal stem cells and the mesenchymal stem cells to be induced are independently pre-screened to obtain the mesenchymal stem cells expressing the surface marker protein CD51. The inventors found that the mesenchymal stem cells expressing the surface marker protein CD51 have a strong ability to differentiate into osteocytes, and thus, through screening (such as flow cytometry screening), to obtain mesenchymal stem cells with a strong ability to differentiate into osteocytes. The mesenchymal stem cells are then pre-induced, and the obtained pre-induced cells are combined with the mesenchymal stem cells expressing the surface marker protein CD51 to obtain bone cells with a higher yield.

The frequency of administration and dosage of the medicament of the present invention can be determined by a number of related factors including the type of disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient and the medicament as the active ingredient type. According to some embodiments of the invention, the daily dose may be divided into 1, 2 or more doses in suitable form to be administered 1, 2 or more times over the entire time period, so long as a therapeutically effective amount is achieved .

The term "treating" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects caused by the disease. "Treatment" as used herein encompasses disease in mammals, particularly humans, including: (a) preventing disease (eg, preventing bone damage) or the occurrence of a disorder in individuals susceptible to but not yet diagnosed with the disease; (b) inhibiting disease, such as retarding disease progression; or (c) alleviating disease, such as reducing symptoms associated with the disease. "Treatment" as used herein encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, alleviate or inhibit a disease in the individual, including, but not limited to, administering to an individual in need thereof a bone containing tissue engineered as described herein.

According to embodiments of the present invention, the medicaments of the present invention may be used in conjunction with conventional treatment methods and/or therapies, or may be used separately from conventional treatment methods and/or therapies. When the agents of the present invention are administered in combination therapy with other agents, they may be administered to an individual sequentially or simultaneously. Alternatively, the medicament of the present invention may comprise a combination of the tissue engineered bone of the present invention, a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient, and other therapeutic or prophylactic drugs known in the art.

Additional aspects and advantages of the present invention will be set forth, in part, from the following description, and in part will be apparent from the following description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and strongly understood from the following description of embodiments taken in conjunction with the accompanying drawings, wherein:

Figure 1 shows a morphological diagram of mesenchymal stem cells according to an embodiment of the present invention;

Fig. 2 shows the expression analysis chart of mesenchymal stem cell surface marker protein according to an embodiment of the present invention; and

FIG. 3 shows an analysis chart of osteogenic differentiation efficiency according to an embodiment of the present invention.

Detailed ways

Embodiments of the present invention are described in detail below. The embodiments described below are exemplary, only for explaining the present invention, and should not be construed as limiting the present invention.

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Example 1 Identification of surface markers of mesenchymal stem cells by flow cytometry

(1) The mesenchymal stem cells (the cell morphology is shown in Figure 1) were cultured in a 10 cm cell culture dish containing serum-free medium (purchased from Sanli Company, China). When the cells grow and reach more than 90% confluence, aspirate the culture medium and rinse the cells once with PBS buffer, add TrypLe Express enzyme to digest the cells, and when the cells become round and fall off the culture plate, rinse the cells with 10% fetal The αMEM medium with bovine serum terminated the digestion, and the cell suspension was collected into a 15 ml centrifuge tube and centrifuged at 1200 rpm for 5 minutes. After the supernatant was discarded, the cells were resuspended in fresh serum-free medium, and subcultured for 3 generations. The cells of the third generation were taken and digested with trypsin.

(2) Put the digested mesenchymal stem cells into a 1.5 mL EP tube, add 1 μl of flow-through antibodies (CD73, CD90, CD34, CD19, CD14, HLA-DR) respectively, and incubate at 4°C for 30 minutes in the dark.

(3) Wash the cells twice with PBS buffer, and then use flow cytometry to detect the expression of mesenchymal stem cell surface markers. The results are shown in Table 1 and Figure 2. It can be seen that the cell phenotype is uniform, and the expression of cells varies between Plasma cell markers CD73, CD90, hematopoietic cell markers CD34, CD14, CD19 are not expressed, and human leukocyte antigen HLA-DR is not expressed, fully in line with the "International Association of Cell Therapy" on the surface markers of mesenchymal stem cells.

Table 1 Flow cytometry identification

Cell phenotype Detection value normal reference value CD73 95.92% >95% CD90 99.99% >95% CD34 0.11% <2% CD19 0.00% <2% CD14 0.00% <2% HLA-DR 0.00% <2%

Example 2 Screening of mesenchymal stem cells expressing CD51

(1) The mesenchymal stem cells were cultured in a 10 cm cell culture dish containing serum-free medium. When the cells grow and reach more than 90% confluence, aspirate the culture medium and rinse the cells once with PBS buffer, add TrypLe Express enzyme to digest the cells, and when the cells become round and fall off the culture plate, rinse the cells with 10% fetal The αMEM medium with bovine serum terminated the digestion, and the cell suspension was collected into a 15 ml centrifuge tube and centrifuged at 1200 rpm for 5 minutes. After the supernatant was discarded, the cells were resuspended in fresh serum-free medium, and subcultured for 3 generations. The cells of the third generation were taken and digested with trypsin.

(2) Put the digested mesenchymal stem cells into a 1.5 mL EP tube, add 10 μl CD51 flow antibody, and incubate at 4° C. for 30 minutes in the dark.

(3) The cells were washed twice with PBS buffer, and then the mesenchymal stem cells expressing CD51 were sorted out by flow cytometer. After detection, the expression level of CD51 in the mesenchymal stem cells was about 4%.

(4) Collect the cells, inoculate them in a 10cm cell culture dish, and then put them into a 37°C, 5% CO ₂ incubator for culture.

Example 3 Comparison of osteogenic differentiation of mesenchymal stem cells + pre-induced cells and mesenchymal stem cells

(1) When the mesenchymal stem cells screened in Example 2 are cultured to 80-90% confluence, the cells are digested with trypsin and counted, and then inoculated into a 6-well plate (containing 10 vol% fetal cells at 8000 cells/cm ² ) bovine serum in α-MEM medium), and then the cells were cultured in a 37°C, 5% _CO2 incubator.

(2) On the second day, the osteogenic induction medium was replaced, and the medium was composed of DMEM, 10% fetal bovine serum, 0.1 mM dexamethasone, 10 mM sodium β-glycerophosphate and 50 μM ascorbic acid.

(3) The medium was changed every two days, and the cells were induced for 7 days, and the obtained cells were pre-induced cells.

(4) After 7 days, aspirate the medium, rinse the cells once with PBS buffer, add TrypLe Express enzyme to digest the cells, and use αMEM medium containing 10% fetal bovine serum in time when the cells become round and fall off the culture plate To terminate the digestion, collect the cell suspension into a 15 ml centrifuge tube and centrifuge at 1200 rpm for 5 minutes. The collected pre-induced cells were 6×10 ⁷ cells/mL, the mesenchymal stem cells screened in Example 2 were 3×10 ⁷ cells/mL, and 2×10 ¹⁰ cells/mL platelet-rich plasma; 1% w/v hydrogel gel; and 100 μM tris(2-dimethylaminoethyl)amine mixed and seeded in a 6-well plate. At the same time, only the mesenchymal stem cells screened in Example 2 were set as the control experimental group.

(5) On the second day, the osteogenic induction medium was replaced with DMEM, 10% fetal bovine serum, 0.1 mM dexamethasone, 10 mM sodium β-glycerophosphate and 50 μM ascorbic acid, and the medium was changed every two days to induce 20 Days later, calcium nodules were formed, alizarin red staining was performed, and the results were observed under a microscope. The results are shown in Figure 3, where A is the control experimental group, containing only mesenchymal stem cells; B is the experimental group, containing mesenchymal stem cells and pre-induced cells. It can be seen that the number of calcium nodules formed by the mesenchymal stem cells and the pre-induced cells group is significantly more than that of the mesenchymal stem cells only group, indicating that the mesenchymal stem cells and the pre-induced cells act together, and the obtained osteoblasts can be obtained. higher rate.

In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples, without conflicting each other.

Although the embodiments of the present invention have been shown and described above, it should be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Embodiments are subject to variations, modifications, substitutions and variations.

Claims (8)

1. the purposes of tissue engineering bone in the preparation of medicine, it is characterised in that the medicine is used for the treatment of bone injury disease, The tissue engineered bone includes: mesenchymal stem cells and pre-induced mesenchymal stem cells; The tissue engineered bone further comprises at least one of the following: 2×10 ¹⁰ /mL platelet-rich plasma; 1% w/v hydrogel; and 100 μM tris(2-dimethylaminoethyl)amine; The number ratio of the mesenchymal stem cells to the pre-induced mesenchymal stem cells is 1:1-4; The pre-induced mesenchymal stem cells are obtained by: contacting the mesenchymal stem cells to be induced with an expansion medium to perform expansion culture, so as to obtain an expansion culture product; and contacting the expanded culture product with a pre-induction medium for pre-induction culture, so as to obtain the pre-induced mesenchymal stem cells; The pre-induction culture time is 6-8 days; The expansion medium comprises: α-MEM medium containing 10% by volume of fetal bovine serum, The pre-induction medium includes: 50 μM ascorbic acid; 10 mM sodium β-glycerophosphate; 0.1 mM dexamethasone; and a basal medium, the basal medium includes: DMEM medium containing 10 vol% fetal bovine serum; The content of the mesenchymal stem cells is 1×10 ⁷ to 1×10 ⁸ cells/mL; The content of the pre-induced mesenchymal stem cells is 1×10 ⁷ to 1×10 ⁸ cells/mL; The mesenchymal stem cells express the surface marker protein CD51. 2. The use according to claim 1, wherein the drug is administered by injection. 3 . The use according to claim 1 , wherein the number ratio of the mesenchymal stem cells to the pre-induced mesenchymal stem cells is 1:2. 4 . The use according to claim 1, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, amniotic fluid stem cells or dental pulp stem cells. The use according to claim 1, wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells. 6 . The use according to claim 1 , wherein contacting the amplified culture product with the pre-induction medium is to make the amplified culture product 3×10 ³ to 8×10 ³ cells/cm ² . The inoculum amount was inoculated in the pre-induction medium. 7. purposes according to claim 1, is characterized in that, contacting described expanded culture product with pre-induction medium is to inoculate described expanded culture product according to the inoculation amount of 5 × 10 ³ / cm ² in the pre-induction medium. 8. The use according to claim 1, wherein the pre-induction culture time is 7 days.

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