脉冲电磁场对脱钙骨基质诱导人骨髓间充质干细胞增殖与成骨分化的影响

发布时间：2018-01-17 02:03

本文关键词：脉冲电磁场对脱钙骨基质诱导人骨髓间充质干细胞增殖与成骨分化的影响　出处：《第三军医大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景和目的:脱钙骨基质(Demineralized Bone Matrix, DBM)具有在体外促进人骨髓间充质干细胞(human Mesenchymal Stem Cells, hMSCs)增殖与成骨分化,在体内促进新骨形成的能力,在临床组织工程中具有广泛应用价值。hMSCs具有良好的成骨细胞分化潜能和成骨活性,是骨组织工程种子细胞的最佳选择之一,hMSCs与DBM共同培养,与DBM释放的可溶性骨生长因子接触后,能加速自身的增殖与成骨分化。脉冲电磁场(pulsed electromagnetic fields, PEMF)在矫形外科领域主要用于治疗骨不连、骨延迟愈合及假关节形成等并已经取得成功。特定频率、特定场强的PEMF能够刺激成骨细胞增殖、分化,并可刺激局部骨生长因子的产生,加速骨基质矿化,该现象已被国内外学者们证实。然而有关PEMF和DBM共同刺激hMSCs增殖与成骨分化的具体生物学作用研究尚未见报道。本研究将观察PEMF对DBM诱导hMSCs在体外培养时细胞增殖与成骨分化的影响,探讨PEMF影响组织工程材料中hMSCs成骨分化的可能机制,为骨组织工程提供新的技术手段。方法: 1.经知情同意后,选取因股骨颈骨折行髋关节置换术患者的股骨头。 2.将股骨头锯成3mm×5mm×5mm大小的骨粒,按Urist b法制作成DBM。用扫描电镜观察材料的形态并测量材料孔径,能谱仪检测DBM钙含量。 3.获取健康志愿者的骨髓,分离、培养hMSCs,进行原代和传代培养。鉴定hMSCs的表面抗原标志物CD44和CD105。 4.第3代hMSCs以1×104个/孔接种至8块24孔板,以1×105个/孔接种至2块加盖玻片的6孔板,各分成4组:细胞对照组(cell, C组)、细胞+材料组(cell-DBM, CD组)、细胞+脉冲电磁场组(cell-PEMF, CP组)、细胞+材料+脉冲电磁场组(cell-DBM-PEMF, CDP组),其中CD组和CDP组培养板每孔放入一块DBM材料,CP组和CDP组给予PEMF(4h/d,频率15Hz,场强5Gs)照射。 5.在第1、3、7、10、14天时用MTT法分析细胞的增殖活性;在第1、7、14、21天时采用ELISA方法检测碱性磷酸酶(alkaline phosphatase, ALP)活性、放免法检测骨钙素(osteocalcin, OC)浓度。 6.在第21天时,进行钙结节茜素红染色,然后在Olympus显微镜(IX71型,日本)下进行组织学观察,并统计钙结节数量。结果: 1.制作的DBM颜色淡黄,质地较软。扫描电镜下可见DBM孔隙表面较光滑,孔隙大小为115.4μm-188.4μm,平均孔径为140.85±21.75μm。能谱仪检测DBM钙含量为11.49±0.84%。 2.分离的原代hMSCs呈圆形,24h后可见细胞贴壁,呈梭形生长;当hMSCs生长达80%融合后,呈同向性生长,旋涡状排列,细胞细长而有突起。hMSCs的表面抗原CD44、CD105免疫组化染色阳性,细胞膜呈棕褐色。 3.根据MTT检测结果,CD组、CP组和CDP组生长曲线较C组前移,CDP组较C组、CD组、CP组有更高的增殖峰值(p 0.01);在第7、10、14天时,CDP组较C组、CD组、CP组升高(P 0.01),在第7、10天时,CD组较CP组升高(P 0.01)。 4.种植后1天,各组间ALP活性无明显差异。在种植7天后CD组、CP组、CDP组ALP活性均明显升高(p 0.01),随即CP组开始持续下降,而14天时CD组和CDP组ALP活性才达到最高值。第7、14天时CDP组ALP活性均较CD组、CP组显著升高(p 0.01),第14天时CD组ALP活性较CP组显著升高(p 0.01)。在21天时CD组、CP组、CDP组组间ALP活性无显著性差异,但以上三组仍较C组显著升高(p 0.01)。 5.除C组外,CD组、CP组和CDP组的OC值在种植7天后均持续显著增高(p 0.01),其中CDP组增速最快,CD组和CP组OC值在前14天培养过程中均无显著性差异,第21天时CD组较CP组显著升高。CDP组OC值在7、14、21天时均较CD组、CP组显著升高(p 0.01)。 6.在21天时钙结节茜素红染色在40倍镜下观察,平均视野内形成的钙结节数目为C组0个,CD组13.50±1.93个,CP组9.88±1.55个,CDP组19.13±3.27,钙结节数量CDP组明显要多于另外三组(p 0.01), CD组多于CP组(p 0.01)。结论: 1. DBM与PEMF联合刺激及DBM和PEMF单独刺激均加快hMSCs的增殖;DBM促hMSCs增殖能力强于PEMF;DBM与PEMF联合刺激hMSCs增殖较DBM和PEMF单独刺激更强。 2. DBM和PEMF单独刺激促进hMSCs成骨分化的时间过程基本一致,但DBM的促分化作用更强;DBM与PEMF联合对hMSCs的促分化作用较DBM和PEMF单独刺激不仅开始时间早,而且强度更大。 3.在DBM和PEMF单独作用促进钙盐沉积的后期,DBM的作用更强;DBM与PEMF联合刺激促进hMSCs成骨分化钙盐沉积时,不仅强度大而且持续时间更长。 4. PEMF对DBM诱导hMSCs增殖与成骨分化具有明显的协同效应,外源性的电磁场可以用于骨组织工程促进种子细胞的分化和成骨。
[Abstract]:Background and objective: the decalcified bone matrix (Demineralized Bone Matrix, DBM) with in vitro promotes human bone marrow mesenchymal stem cells (human Mesenchymal Stem Cells, hMSCs) proliferation and osteogenic differentiation in vivo, promote new bone formation, in clinical tissue engineering has wide application value with.HMSCs cell differentiation into bone good and osteogenic activity, is one of the best choice of seed cells for bone tissue engineering, co culture of hMSCs and DBM, growth factor and soluble bone contact DBM release, can accelerate the proliferation and osteogenic differentiation.
Pulsed electromagnetic fields (pulsed electromagnetic fields, PEMF) in the field of orthopaedic surgery is mainly used for the treatment of bone nonunion, bone nonunion and pseudarthrosis and have been successful. Specific frequency, specific field PEMF can stimulate proliferation and differentiation of bone cells, and can stimulate local production of bone growth factors, accelerate the mineralization of bone matrix. This phenomenon has been confirmed by domestic and foreign scholars. However, the specific biological function of research on PEMF and DBM stimulated hMSCs proliferation and osteogenic differentiation has not been reported.
This study will observe the effect of PEMF on the proliferation and osteogenic differentiation of DBM induced hMSCs in vitro, and explore the possible mechanism of PEMF affecting the osteogenic differentiation of hMSCs in tissue-engineered materials, so as to provide new technical means for bone tissue engineering.
Method:
1. after informed consent, the femoral head was selected for the hip arthroplasty for the femoral neck fracture.
2., the femoral head was sawed into the size of 3mm * 5mm * 5mm. The DBM. was made according to Urist B method. The morphology of the material was observed by scanning electron microscope, and the pore diameter of the material was measured. The calcium content of DBM was detected by EDS.
3. obtain the bone marrow of healthy volunteers, separate, culture hMSCs, carry out primary and subculture. Identify the surface antigen markers of hMSCs, CD44 and CD105.
The third generation of hMSCs 4. with a 1 x 104 / 8 holes inoculated to 24 well plates with 1 * 105 / 6 hole plate hole inoculation to 2 pieces with glass, each divided into 4 groups: control group (cell cells, C cells + group), material group (cell-DBM, CD), cell + pulse electromagnetic field group (cell-PEMF, group CP), cells + materials + pulsed electromagnetic field group (cell-DBM-PEMF, group CDP), CD group and CDP group were cultured in each hole into a piece of material DBM, CP group and CDP group were given PEMF (4h/d, frequency 15Hz, field 5Gs) irradiation.
5. on the day of 1,3,7,10,14, the proliferative activity of cells was analyzed by MTT method. The activity of alkaline phosphatase (alkaline phosphatase ALP) was detected by ELISA on day 1,7,14,21, and the concentration of osteocalcin (osteocalcin, OC) was detected by radioimmunoassay.
6. at twenty-first days, calcium nodules were stained with alizarin red, then the histological observation was performed under the Olympus microscope (IX71, Japan), and the number of calcium nodules was counted.
Result:
The color of DBM made of 1. is light yellow and soft. Under scanning electron microscope, the DBM pore surface is smoother, the pore size is 115.4 m-188.4 m-188.4 m, and the average pore size is 140.85 + 21.75 m.. The DBM content of DBM is 11.49 + 0.84%. by EDS.
2., the primary hMSCs of the isolate was round. After 24h, the cells adhered to the wall and grew in spindle shape. When the hMSCs growth reached 80% fusion, the cells grew in the same direction, whirlpool arranged, the cells were elongated and the surface antigen CD44 of.HMSCs was raised, CD105 was positive for immunohistochemical staining, and the cell membrane was brown.
3. according to the results of MTT test, the growth curve of group CD, group CP and group CDP moved forward compared with C group. The CDP group had a higher peak value of proliferation than the C group, CD group and CP group (P 0.01). On the first day of 7,10,14, the group of CP increased (0.01), and the level of group a increased (CP 0.01).
1 4. days after planting, there was no significant difference between the activity of ALP in 7 days after planting. CD group, CP group, CDP group, ALP activity increased significantly (P 0.01), and group CP continued to decline, and 14 days in CD group and CDP group ALP activity was reached the highest value of the first 7,14 days CDP. The activity of ALP were lower than CD group, CP group increased significantly (P 0.01), CD in ALP group was significantly higher than that of CP group at day fourteenth (P 0.01). On the 21 day in CD group, CP group, CDP group has no significant difference between the groups of ALP activity, but more than three were still significantly compared with C group increased (P 0.01).
5. except for C group, CD group, CP group and CDP group OC values at 7 days after planting were significantly higher (P 0.01), the CDP group has the fastest growth rate, CD group and CP group OC in the 14 days before the train had no significant difference during the twenty-first day CD group than in CP the value of OC increased significantly in group.CDP group in 7,14,21 days when compared with CD group, CP group increased significantly (P 0.01).
6., on the 21 day, calcium nodules and alizarin red staining were observed under 40 times microscope. The number of calcium nodules formed in the average field was 0 in group C, 13.50 in group CD, 1.93 in group CD, 9.88 in group CP, 1.55 in group CP, 19.13 19.13 in 3.27 group, and the number of calcium nodules in CDP group was significantly more than that in other three group (P three), and CD group was more than CP group (P P).
Conclusion:
1., the combined stimulation of DBM and PEMF and DBM and PEMF alone stimulated the proliferation of hMSCs, DBM promoted hMSCs proliferation ability stronger than PEMF, and DBM and PEMF stimulated the proliferation of hMSCs more strongly than those of both DBM and H.
2., the time course of promoting hMSCs osteogenesis by DBM and PEMF alone is basically the same, but DBM promotes differentiation. The combination of DBM and PEMF promotes the differentiation of hMSCs more than that of DBM and PEMF alone.
3., in the late stage of DBM and PEMF promoting calcium deposition alone, DBM plays a more powerful role. The joint stimulation of DBM and PEMF promotes hMSCs osteogenesis and calcium deposition.
4. PEMF has obvious synergistic effect on DBM induced hMSCs proliferation and osteogenic differentiation. Exogenous electromagnetic fields can be used in bone tissue engineering to promote differentiation and osteogenesis of seed cells.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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