CN103667182A - Inducing method and inducing culture medium for differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitro - Google Patents
- ️Wed Mar 26 2014
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- CN103667182A CN103667182A CN201310514265.9A CN201310514265A CN103667182A CN 103667182 A CN103667182 A CN 103667182A CN 201310514265 A CN201310514265 A CN 201310514265A CN 103667182 A CN103667182 A CN 103667182A Authority
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Abstract
本发明提供了一种骨髓间充质干细胞在体外向成骨细胞分化的诱导方法及诱导培养基，本发明诱导培养基组成为：地塞米松1×10-8mol/L，抗坏血酸50μmol/L，β-甘油磷酸钠10mmol/L，溶剂为骨片完全培养基的上清液，其成分为：胎牛血清10%，青霉素100U/mL，链霉素100mg/L，DMEM培养液和F12培养液的混合物以及骨片培养过程中骨细胞分泌的多种生长因子。本发明通过更换细胞培养液和细胞贴壁传代的方法纯化小鼠骨髓间充质干细胞，取获得的第1代细胞进行诱导，以培养72-96小时的骨片完全培养基的上清液为成骨细胞分化诱导剂的溶剂，明显提高了骨髓间充质干细胞的体外成骨分化效率。
The invention provides a method for inducing bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro and an induction medium. The induction medium of the invention is composed of: dexamethasone 1×10 -8 mol/L, ascorbic acid 50 μmol/L , β-glycerophosphate sodium 10mmol/L, the solvent is the supernatant of bone slice complete culture medium, and its components are: fetal bovine serum 10%, penicillin 100U/mL, streptomycin 100mg/L, DMEM medium and F12 culture The mixture of liquid and various growth factors secreted by bone cells during bone slice culture. The present invention purifies mouse bone marrow mesenchymal stem cells by replacing the cell culture medium and cell adherent subculture, takes the obtained first-generation cells for induction, and uses the supernatant of the complete culture medium of bone slices cultured for 72-96 hours as The solvent of the osteoblast differentiation inducer can obviously improve the osteogenic differentiation efficiency of bone marrow mesenchymal stem cells in vitro.
Description
技术领域 technical field
本发明涉及骨髓间充质干细胞的体外成骨细胞诱导分化方法，以及所用的诱导培养基。 The invention relates to an in vitro osteoblast induction and differentiation method of bone marrow mesenchymal stem cells and the used induction medium.
背景技术 Background technique
骨髓间充质干细胞在适当的条件刺激下可分化为成骨细胞、软骨细胞、脂肪细胞等细胞类型，而且易于分离培养，具有较强的自我更新、分化能力和免疫抑制等优点，使其在组织器官缺损性疾病、组织器官退行性疾病以及细胞治疗和组织工程领域具有重要的应用前景。近来有众多的国内外研究探索骨髓间充质干细胞向成骨细胞诱导分化的方法，以期作为骨组织工程的研究基础和临床骨相关疾病生物治疗的理论指导。体外分离纯化的骨髓间充质干细胞经过传代后，相关的生物学特性发生改变，外形未见明显变化，但是已逐渐失去其分化潜能，经过定向诱导分化后只有少量的细胞向所需的方向分化，影响其作为组织工程细胞来源的应用。近来有研究报道将一些与成骨细胞分化相关的细胞因子应用于干细胞成骨分化相关的研究中，对提高骨髓间充质干细胞的成骨分化效率具有一定的生物学效应。 Bone marrow mesenchymal stem cells can be differentiated into osteoblasts, chondrocytes, adipocytes and other cell types under the stimulation of appropriate conditions, and are easy to isolate and culture, and have strong self-renewal, differentiation ability and immunosuppressive advantages. It has important application prospects in the fields of tissue and organ defect diseases, tissue and organ degenerative diseases, cell therapy and tissue engineering. Recently, many domestic and foreign studies have explored the method of inducing the differentiation of bone marrow mesenchymal stem cells into osteoblasts, with a view to serving as the research basis of bone tissue engineering and the theoretical guidance of clinical biological treatment of bone-related diseases. After the bone marrow mesenchymal stem cells isolated and purified in vitro have been passaged, the relevant biological characteristics have changed, and the appearance has not changed significantly, but they have gradually lost their differentiation potential. After directional induction and differentiation, only a small number of cells differentiate in the desired direction , affecting its application as a source of tissue engineering cells. Recently, it has been reported that some cytokines related to osteoblast differentiation are applied to the research related to osteogenic differentiation of stem cells, which has a certain biological effect on improving the osteogenic differentiation efficiency of bone marrow mesenchymal stem cells.
发明内容 Contents of the invention
本发明所要解决的技术问题是提供一种骨髓间充质干细胞在体外向成骨细胞分化的诱导方法，能提高分化效率。 The technical problem to be solved by the present invention is to provide a method for inducing bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro, which can improve the differentiation efficiency.
本发明解决技术问题所采用的技术方案是： The technical scheme that the present invention solves technical problem adopts is:
一种骨髓间充质干细胞在体外向成骨细胞分化的诱导方法，所述方法包括： A method for inducing bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro, the method comprising:
（一）骨髓间充质干细胞的体外分离、培养和纯化 (1) In vitro isolation, culture and purification of bone marrow mesenchymal stem cells
取骨髓细胞，经筛网过滤后制成单细胞悬液接种于培养皿中进行贴壁培养，初始接种密度为5×106个/mL细胞培养基，培养3小时后更换细胞培养基（组成同前），此后每隔12小时更换一次细胞培养基（组成同前），直到接种后72小时，然后再每隔3天更换细胞培养基（组成同前）继续培养，直到细胞长至80%~90%融合，以胰酶消化，消化时间不超过2分钟，细胞传代后继续培养或直接接种于孔板中用于诱导向成骨细胞分化；所述细胞完全培养基终浓度组成如下：胎牛血清10%（体积百分比浓度），青霉素100U/mL，链霉素100mg/L，溶剂为基础培养基，所述基础培养基为DMEM培养液和F12培养液体积比为1:1的混合物。 Bone marrow cells were collected and filtered through a sieve to make a single-cell suspension and inoculated in a culture dish for adherent culture. The initial inoculation density was 5× 106 /mL cell culture medium, and the cell culture medium was replaced after 3 hours of culture (composition The same as before), then replace the cell culture medium (same composition as before) every 12 hours thereafter until 72 hours after inoculation, and then replace the cell culture medium (same composition as before) every 3 days to continue culturing until the cells grow to 80% ~90% fusion, digested with trypsin, the digestion time is not more than 2 minutes, continue to culture after cell subculture or directly inoculated in the well plate to induce differentiation into osteoblasts; the final concentration of the complete culture medium of the cells is composed as follows: Bovine serum 10% (volume percentage concentration), penicillin 100U/mL, streptomycin 100mg/L, the solvent is the basal medium, and the basal medium is a mixture of DMEM culture medium and F12 culture medium with a volume ratio of 1:1.
（二） 骨贴片培养和培养基上清的获取、保存 (2) Bone patch culture and acquisition and storage of medium supernatant
采用无菌的显微钳将已经取出骨髓内容物的小鼠股骨和胫骨分成约4mm2大小的骨片，均匀放置于培养皿中，骨髓腔面接触培养皿底，置于37℃、饱和湿度、含5% CO2的培养箱中进行骨片培养，待培养至72~96小时有大量细胞从骨片爬出时收集其上清液，离心后冻存于－20℃。所述骨片完全培养液终浓度组成如下：胎牛血清10%，青霉素100U/mL，链霉素100mg/L，溶剂为基础培养基，所述基础培养基为DMEM培养液和F12培养液体积比1:1的混合液。 Use sterile microscopic forceps to divide the femur and tibia of the mouse from which the bone marrow content has been removed into bone pieces of about 4 mm 2 in size, and place them evenly in a petri dish, with the bone marrow cavity surface touching the bottom of the petri dish, and place at 37°C and saturated humidity , in an incubator containing 5% CO 2 for bone slice culture, after 72 to 96 hours of culture, when a large number of cells crawled out of the bone slice, collect the supernatant, centrifuge and freeze at -20°C. The final concentration of the complete culture solution of the bone slice is composed as follows: 10% of fetal bovine serum, 100 U/mL of penicillin, 100 mg/L of streptomycin, and the solvent is the basal medium, and the basal medium is DMEM nutrient solution and F12 nutrient solution volume 1:1 mixture.
（三） 骨髓间充质干细胞的成骨诱导分化 (3) Osteogenic differentiation of bone marrow mesenchymal stem cells
原代接种的第1代骨髓间充质干细胞长至80％~90％融合时，添加0.25%胰酶（含有0.02%EDTA）室温消化细胞2分钟，添加完全培养基终止消化后经过离心分离获得细胞沉淀，然后以相同的密度分接种于12孔板中，待细胞扩增至80％融合后添加诱导培养基进行诱导分化。 When the primary inoculated first-generation bone marrow mesenchymal stem cells grow to 80%~90% confluent, add 0.25% trypsin (containing 0.02% EDTA) to digest the cells at room temperature for 2 minutes, add complete medium to stop the digestion, and then centrifuge to obtain The cells were precipitated, and then seeded in 12-well plates at the same density. After the cells expanded to 80% confluence, induction medium was added to induce differentiation.
本发明所要解决的另一个技术问题是提供一种用于骨髓间充质干细胞的体外诱导向成骨分化的诱导培养基，所述诱导培养基的终浓度如下：地塞米松1×10-8 mol/L，抗坏血酸50μmol/L，甘油磷酸钠10 mmol/L，溶剂为所述骨片培养72～96小时的上清液。 Another technical problem to be solved by the present invention is to provide an induction medium for in vitro induction of osteogenic differentiation of bone marrow mesenchymal stem cells, the final concentration of the induction medium is as follows: dexamethasone 1×10 -8 mol/L, ascorbic acid 50 μmol/L, sodium glycerophosphate 10 mmol/L, and the solvent is the supernatant of the bone slices cultured for 72-96 hours.
优选的，所述诱导培养基终浓度组成如下：地塞米松1×10-8 mol/L，抗坏血酸50μmol/L，β-甘油磷酸钠10 mmol/L，溶剂为所述骨片培养72小时的上清液，所述上清液为骨片完全培养液以及在骨片培养过程中骨细胞分泌的多种细胞因子。 Preferably, the composition of the final concentration of the induction medium is as follows: dexamethasone 1×10 -8 mol/L, ascorbic acid 50 μmol/L, β-sodium glycerophosphate 10 mmol/L, and the solvent is the bone slice cultured for 72 hours. Supernatant, the supernatant is the complete culture solution of bone slices and various cytokines secreted by bone cells during the culture process of bone slices.
本发明在以往的研究基础上，采用骨片培养上清液和成骨诱导培养基结合的方法，取传代后的第1代骨髓间充质干细胞进行成骨细胞分化，明显提高了骨髓间充质干细胞的成骨分化效率。 On the basis of previous studies, the present invention adopts the method of combining bone slice culture supernatant with osteogenic induction medium, and takes the passaged first-generation bone marrow mesenchymal stem cells for osteoblast differentiation, which significantly improves bone marrow mesenchymal stem cells. Osteogenic differentiation efficiency of stromal stem cells.
本发明的骨髓间充质干细胞在体外向成骨细胞分化的诱导方法，具有如下优点： The method for inducing bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro has the following advantages:
（1）早期诱导可充分体现骨髓间充质干细胞的分化能力。 (1) Early induction can fully reflect the differentiation ability of bone marrow mesenchymal stem cells.
（2）骨片培养过程中释放到培养液中的多种细胞因子不仅有利于骨髓间充质干细胞的存活，还有利于纯化的骨髓间充质干细胞向成骨细胞方向分化。 (2) A variety of cytokines released into the culture medium during the culture of bone slices are not only beneficial to the survival of bone marrow mesenchymal stem cells, but also conducive to the differentiation of purified bone marrow mesenchymal stem cells into osteoblasts.
（3）骨片培养上清液来源方便，获取方法简单可行。 (3) The source of culture supernatant of bone slices is convenient, and the method of obtaining it is simple and feasible.
附图说明 Description of drawings
图1 为实施例1的成骨细胞分化染色结果图。 Figure 1 is a diagram of the staining results of osteoblast differentiation in Example 1.
图2 为实施例2的成骨细胞分化染色结果图。 Figure 2 is a diagram of the staining results of osteoblast differentiation in Example 2.
图3 为实施例3的成骨细胞分化染色结果图。 Figure 3 is a diagram of the staining results of osteoblast differentiation in Example 3.
图4为骨片培养至72小时的细胞爬片图。 Fig. 4 is the cell creeping picture of bone slice cultured to 72 hours.
其中，图中箭头所示为钙结节沉淀，红色为钙化物沉淀，图片的放大倍率均为100倍。 Among them, the arrows in the figure show the calcium nodule precipitation, and the red color is the calcification precipitation, and the magnification of the pictures is 100 times.
具体实施方式 Detailed ways
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。 The present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto.
实施例1： Example 1:
所用试剂：胎牛血清（Hyclone），青霉素、链霉素、DMEM培养液、F12培养液（Gibco），地塞米松、抗坏血酸、甘油磷酸钠、5’－氮胞苷（Sigma）。 Reagents used: fetal bovine serum (Hyclone), penicillin, streptomycin, DMEM medium, F12 medium (Gibco), dexamethasone, ascorbic acid, sodium glycerophosphate, 5'-azacytidine (Sigma).
1、取6周左右的ICR雄性成年小鼠（购买于浙江省医学科学院实验动物中心），颈椎脱臼处死，无菌条件下取出股骨和胫骨。 1. Take about 6-week-old ICR male adult mice (purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences), kill them by cervical dislocation, and take out the femur and tibia under aseptic conditions.
2、无菌剪刀剪开两端骨骺，用注射器吸取完全培养基冲洗骨髓腔，完全培养基组份：10%（v/v）胎牛血清，青霉素100U/mL，链霉素100mg/L，溶剂为DMEM/F12培养液（即DMEM与F12体积比1:1的混合液）。 2. Cut the epiphyses at both ends with sterile scissors, and use a syringe to draw complete medium to flush the bone marrow cavity. Complete medium components: 10% (v/v) fetal bovine serum, penicillin 100U/mL, streptomycin 100mg/L, The solvent is DMEM/F12 culture medium (that is, a mixture of DMEM and F12 with a volume ratio of 1:1).
3、获得的骨髓细胞悬液经200目筛网过滤，接种于10 cm的培养皿中，初始接种密度约为5×106个/mL，细胞培养基为完全培养基，初始接种的细胞培养基体积为2mL，可覆盖培养皿底。 3. The obtained bone marrow cell suspension was filtered through a 200-mesh sieve, and inoculated in a 10 cm culture dish with an initial inoculation density of about 5×10 6 cells/mL. The cell culture medium was complete medium, and the initial inoculated cell culture The base volume is 2mL, which can cover the bottom of the petri dish.
4、初始接种的骨髓细胞置于37℃、饱和湿度、含5% CO2的培养箱中培养3小时后更换细胞培养基，体积为2 mL。 4. The initially inoculated bone marrow cells were cultured in an incubator at 37°C, saturated humidity, and 5% CO 2 for 3 hours, and then the cell culture medium was replaced with a volume of 2 mL.
5、初次更换培养基12小时后，再次更换细胞培养基，体积仍为2 mL，12小时后，再次更换细胞培养基，至初次接种后72小时，更换细胞培养基体积增加为8 mL，之后每3天更换一次细胞培养基，体积为8 mL，待细胞长至80%融合时进行消化传代接种于12孔板中，在倒置显微镜下观察培养不同时间的细胞形态。 5. 12 hours after the initial replacement of the medium, replace the cell culture medium again, the volume is still 2 mL, 12 hours later, replace the cell culture medium again, until 72 hours after the initial inoculation, the volume of the replacement cell culture medium is increased to 8 mL, after that The cell culture medium was replaced every 3 days, with a volume of 8 mL. When the cells reached 80% confluence, they were digested and subcultured in a 12-well plate, and the cell morphology at different times of culture was observed under an inverted microscope.
6、收集骨髓细胞的同时，用显微钳将已经取出骨髓内容物的股骨和胫骨用分为约4mm2的骨片，骨髓腔面向下均匀放置于30mm的培养皿中，每只小鼠的骨片放置于同一个培养皿中培养，每个培养皿中添加的完全培养液体积为2mL，置于37℃、饱和湿度、含5% CO2的培养箱中培养72小时，骨片周围有大量细胞爬出时（如图4所示），吸取上清液至离心管中，10000转/分钟离心5分钟，收集上清液，冻存于－20℃备用（收集的上清液即为步骤6中成骨诱导分化所用的骨片培养上清液）。 6. While collecting bone marrow cells, divide the femur and tibia from which the bone marrow content has been taken out into bone pieces of about 4mm2 with microscopic forceps, and place the bone marrow cavity side down evenly in a 30mm culture dish. The bone slices were cultured in the same petri dish, and the volume of complete culture solution added to each petri dish was 2mL, and cultured in an incubator at 37°C, saturated humidity, and 5% CO 2 for 72 hours. When a large number of cells crawled out (as shown in Figure 4), draw the supernatant into a centrifuge tube, centrifuge at 10,000 rpm for 5 minutes, collect the supernatant, and store it at -20°C for later use (the collected supernatant is The bone slice culture supernatant used for osteogenic differentiation in step 6).
7、细成骨诱导分化：接种于12孔板中的第1代细胞长至80%融合时，PBS缓冲液洗涤细胞三次后，添加0.25%（w/v）胰酶（含有0.02%（w/v）EDTA）室温消化细胞2 min，细胞完全培养基终止消化，细胞悬液经300g离心5 min后，完全培养基悬浮细胞接种于12孔板中继续培养，待细胞长至80％融合时，更换为骨片培养上清液，添加地塞米松1×10-8 mol/L、抗坏血酸50μmol/L、β-甘油磷酸钠10 mmol/L后进行成骨诱导分化，每3天更换一次诱导培养基，分化21天后采用茜素红S染色检测小鼠骨髓间充质干细胞的成骨细胞分化结果，所述诱导培养基的终浓度组成为：地塞米松1×10-8 mol/L，抗坏血酸50μmol/L，β-甘油磷酸钠10 mmol/L，溶剂为培养72小时的骨片培养上清液。 7. Osteogenic induction and differentiation: When the first passage cells seeded in 12-well plates grow to 80% confluence, wash the cells three times with PBS buffer, add 0.25% (w/v) trypsin (containing 0.02% (w/v) /v) EDTA) Digest the cells at room temperature for 2 minutes, stop the digestion with the complete medium of the cells, centrifuge the cell suspension at 300g for 5 minutes, inoculate the suspended cells in the complete medium in a 12-well plate and continue to culture until the cells grow to 80% confluent , replaced with bone slice culture supernatant, added dexamethasone 1×10 -8 mol/L, ascorbic acid 50 μmol/L, β-glycerophosphate sodium 10 mmol/L, and then osteogenic differentiation was performed, and the induced differentiation was performed every 3 days. Culture medium, Alizarin red S staining was used to detect the osteoblast differentiation results of mouse bone marrow mesenchymal stem cells after 21 days of differentiation, and the final concentration of the induction medium was composed of: dexamethasone 1×10-8 mol/L, Ascorbic acid was 50 μmol/L, sodium β-glycerophosphate was 10 mmol/L, and the solvent was culture supernatant of bone slices cultured for 72 hours.
实施例2： Example 2:
本实施例步骤7中进行诱导分化的的骨髓间充质干细胞是通过细胞贴壁培养和胰酶消化传至第3代的细胞，本实施例的其他步骤与实施例1相同。 The bone marrow mesenchymal stem cells induced to differentiate in step 7 of this embodiment are cells that have been passed to the third passage through cell adherent culture and trypsinization, and other steps of this embodiment are the same as those of embodiment 1.
实施例3： Example 3:
本实施例中的诱导培养基的成分是细胞完全培养基添加与实施例1中相同浓度的地塞米松、抗坏血酸和β-甘油磷酸钠。 The composition of the induction medium in this example is that the complete cell medium is supplemented with the same concentrations of dexamethasone, ascorbic acid and sodium β-glycerophosphate as in Example 1.
骨髓间充质干细胞的成骨细胞诱导分化结果鉴定： Identification of osteoblast-induced differentiation of bone marrow mesenchymal stem cells:
实施例1和实施例2、3中的小鼠骨髓间充质干细胞成骨诱导分化21天后，PBS洗涤细胞，4％多聚甲醛固定30分钟，蒸馏水洗涤3遍，0.2％的茜素红S室温染色30分钟，然后弃去染色液，蒸馏水洗涤3遍，显微镜下观察小鼠骨髓间充质干细胞的成骨细胞分化结果。 After 21 days of osteogenic induction and differentiation of mouse bone marrow mesenchymal stem cells in Example 1 and Example 2, 3, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed 3 times with distilled water, and treated with 0.2% Alizarin Red S Stain at room temperature for 30 minutes, then discard the staining solution, wash with distilled water three times, and observe the osteoblast differentiation results of mouse bone marrow mesenchymal stem cells under a microscope.
结果如附图，图1为实施例1的成骨分化细胞茜素红S染色结果显示细胞密度较高，视野中几乎所有的细胞均染成红色，且有众多的钙化物和钙结节沉淀（图1所示），分化效果明显；实施例2采用传至第3代的细胞进行成骨诱导分化，只有少量的钙结节形成，而且细胞已经失去原来的形态，变得宽大扁平，细胞界限模糊（图2所示）；实施例3采用细胞完全培养基添加诱导剂各成分对第1代细胞进行成骨诱导，经过21天的诱导后，细胞密度相对较低，有钙化物和钙结节沉淀形成（图3所示），显然实施例1中的分化效果好，分化效率明显提高。 The results are shown in the accompanying drawings. Figure 1 shows the results of Alizarin Red S staining of the osteogenic differentiated cells in Example 1. It shows that the cell density is high, and almost all the cells in the field of view are stained red, and there are many calcifications and calcium nodules. (As shown in Figure 1), the differentiation effect is obvious; in Example 2, cells passed to the third generation were used for osteogenic differentiation, only a small amount of calcium nodules were formed, and the cells had lost their original shape and became wide and flat. The boundaries are blurred (as shown in Figure 2); in Example 3, the first generation cells were induced to osteogenesis by adding the components of the inducer to the complete cell medium. After 21 days of induction, the cell density was relatively low, with calcification and calcium The formation of nodule precipitates (as shown in Figure 3), obviously, the differentiation effect in Example 1 is good, and the differentiation efficiency is obviously improved.
Claims (6)
1.一种骨髓间充质干细胞在体外向成骨细胞分化的诱导方法，其特征在于：所述方法包括：获取骨髓细胞制成单细胞悬液接种于培养皿中进行贴壁培养，培养3~5小时后首次更换细胞培养基，此后每隔12小时再次更换细胞培养基，直到接种后72小时，然后再每隔3天更换细胞培养基继续培养，直到细胞长至80%~90%融合，以胰酶消化，消化时间不超过2分钟，获得纯化骨髓间充质干细胞； 1. A method for inducing bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro, characterized in that: the method comprises: obtaining bone marrow cells to make a single cell suspension and inoculating them in a culture dish for adherent culture, culturing for 3 Change the cell culture medium for the first time after ~5 hours, then change the cell culture medium again every 12 hours until 72 hours after seeding, and then change the cell culture medium every 3 days to continue the culture until the cells grow to 80%~90% confluent , digested with trypsin, the digestion time is not more than 2 minutes, to obtain purified bone marrow mesenchymal stem cells; 取小鼠的股骨和胫骨进行骨片培养，培养至72~96小时有细胞爬出时，收集骨片培养的上清液； Take the femur and tibia of the mouse for bone chip culture, and when the cells crawl out after 72-96 hours, collect the supernatant of the bone chip culture; 取纯化骨髓间充质干细胞进行体外成骨细胞的诱导分化，每3天更换一次诱导培养基，至诱导21天进行染色鉴定；所述诱导培养基的终浓度组成如下：地塞米松1×10-8 mol/L，抗坏血酸50μmol/L，β-甘油磷酸钠10 mmol/L，溶剂为所述骨片培养72~96小时的上清液。 Purified bone marrow mesenchymal stem cells were used to induce differentiation of osteoblasts in vitro, and the induction medium was replaced every 3 days until 21 days after induction for staining and identification; the final concentration of the induction medium was as follows: dexamethasone 1×10 -8 mol/L, ascorbic acid 50 μmol/L, β-sodium glycerophosphate 10 mmol/L, and the solvent is the supernatant of the bone slices cultured for 72-96 hours. 2.如权利要求1所述的一种骨髓间充质干细胞在体外向成骨细胞分化的诱导方法，其特征在于：所述细胞培养基的终浓度组成如下：胎牛血清10%，青霉素100U/mL，链霉素100mg/L，溶剂为基础培养基；所述基础培养基为DMEM培养液和F12培养液体积比1:1的混合液。 2. A method for inducing bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro, characterized in that: the final concentration of the cell culture medium is composed as follows: fetal bovine serum 10%, penicillin 100U /mL, streptomycin 100mg/L, and the solvent is the basal medium; the basal medium is a mixture of DMEM culture solution and F12 culture solution with a volume ratio of 1:1. 3.如权利要求1所述的一种骨髓间充质干细胞在体外向成骨细胞分化的诱导方法，其特征在于：所述骨片培养基的终浓度组成如下：胎牛血清10%，青霉素100U/mL，链霉素100mg/L，溶剂为基础培养基；所述基础培养基为DMEM培养液和F12培养液体积比1:1的混合液。 3. A method for inducing bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro as claimed in claim 1, characterized in that: the final concentration of the bone slice medium consists of the following: 10% fetal bovine serum, penicillin 100U/mL, streptomycin 100mg/L, the solvent is the basal medium; the basal medium is a mixture of DMEM culture solution and F12 culture solution with a volume ratio of 1:1. 4.如权利要求1所述的一种骨髓间充质干细胞在体外向成骨细胞分化的诱导方法，其特征在于：所述纯化骨髓间充质干细胞为传代后的第1代细胞。 4 . The method for inducing differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitro according to claim 1 , wherein the purified bone marrow mesenchymal stem cells are the first passage cells after passaging. 5.一种骨髓间充质干细胞在体外向成骨细胞分化的诱导培养基，其特征在于：所述诱导培养基的终浓度组成如下：地塞米松1×10-8 mol/L，抗坏血酸50μmol/L，β-甘油磷酸钠10 mmol/L，溶剂为骨片培养72~96小时的上清液。 5. An induction medium for bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro, characterized in that: the final concentration of the induction medium is composed as follows: dexamethasone 1×10-8 mol/L, ascorbic acid 50 μmol /L, β-glycerophosphate sodium 10 mmol/L, the solvent is the supernatant of bone slices cultured for 72-96 hours. 6.如权利要求5所述的一种骨髓间充质干细胞在体外向成骨细胞分化的诱导培养基，其特征在于：所述成骨细胞分化培养基终浓度组成如下：地塞米松1×10-8 mol/L，抗坏血酸50μmol/L，β-甘油磷酸钠10 mmol/L，溶剂为骨片培养72小时的上清液。 6. The induction medium for bone marrow mesenchymal stem cells to differentiate into osteoblasts in vitro according to claim 5, characterized in that: the final concentration of the osteoblast differentiation medium is composed as follows: dexamethasone 1× 10-8 mol/L, ascorbic acid 50 μmol/L, β-sodium glycerophosphate 10 mmol/L, and the solvent was the supernatant of bone slices cultured for 72 hours.
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