大鼠雪旺细胞与成骨细胞联合培养构建神经化组织工程骨的前期研究

发布时间：2018-07-09 16:08

本文选题：骨组织工程 + 增殖　；参考：《南方医科大学》2010年博士论文

【摘要】：目的 1.体外原代培养SD大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs)、GFP转染骨髓间充质干细胞(GFP-BMSCs)、成骨细胞(osteoblasts, OB)、雪旺细胞(Schwann cells,SCs)、感觉神经元(dorsal root ganglion,DRG)、交感神经元(superior cervical ganglion, SCG)细胞。观察DRG、SCG、SCs对GFP-BMSCs分化的成骨细胞增殖的影响。SCs对BMSCs分化OB及颅骨来源OB增殖的影响及机制研究。 2.研究采用荧光微球655 (Quantum dot 655, QD655)标记大鼠BMSCs体外可行性研究。探讨此染料对大鼠BMSCs的毒性、增殖、成骨分化、动态标记率等功能,为体内示踪研究打下研究基础。 3.研究胶原-生物活性玻璃材料的生物相容性研究,包括溶血试验,浸提液对RSC96细胞株增殖试验,胶原-生物活性玻璃干湿态下力学强度研究,最佳接种液体量研究,最佳接种方式方式研究、BMSCs与SCs粘附于材料增殖研究,材料对细胞促增殖试验及向成骨促分化试验,以及初步体内皮下包埋验证成骨试验。 4.探讨采用克氏针与快速成型技术制成接骨板固定大鼠8mm骨缺损稳定性研究,采用体内轴向压缩生物力学测试及体内动态观察固定失效率进行试验,探求两种固定方式的方法、手术技巧、优点及缺点,探求合适固定大鼠骨缺损固定方式。 5.探讨采用大鼠隐动静脉及隐神经束作为神经血管束移植作为外源性因素同步构建神经血管化组织工程骨的可行性,以及采用荧光微球标记的SCs与GFP转染的成骨细胞联合负载于胶原-生物活性玻璃体内示踪可行性研究。方法 1.以2周龄SD大鼠为试验动物模型,采用股骨、胫骨骨髓冲洗法收集细胞,以培养的第3代BMSCs用于试验；采用1d龄乳鼠2只,采用75%酒精浸泡15min,无菌取出颅骨,采用无菌眼科剪建成1-2mm3碎片。向剪碎的骨组织中加入1mL0.25%的胰蛋白酶37℃,消化90min,稍离,去上清。加入0.2%Ⅰ型胶原酶3-5mL,37℃,连续消化2次60min。中止消化,收集细胞,重复上述试验,收集细胞,计数后,种植于25 cm2培养瓶中,置于37℃、5%CO2恒温培养箱中培养。24h后换液,以后每2-3d换液1次。达到80%-90%融合时(原代培养3-5d),消化传代,分组培养,采用碱性磷酸酶染色进行鉴定,2-5代用于试验；采用15d孕鼠,无菌取出胎鼠,取背根神经节,0.25%胰酶消化50min,种植到预先铺板的盖玻片上,进行感觉神经元培养；采用1d龄乳鼠,取颈上神经节,0.25%胰酶消化30min,种植到预先铺板的培养板；取1d乳鼠,取出坐骨神经及臂丛神经,采用组织块贴壁法获得SCs。采用P3 BMSCs向成骨诱导分化2周,进行鉴定；分别采用SCs、DRG、SCG与颅骨来源成骨细胞和BMSCs来源成骨细胞共培养检测,检测共培养后成骨细胞增殖及分化机制。 2.采用QD655按照试剂盒推荐浓度,分别将QD试剂A和试剂载体B各取1μL混置于1.5mL微量离心管中,放于孵育15min,加入100gL培养基,涡旋30s,向管中加入含106个P3细胞的0.2mL悬液,打匀后置于37℃、5%CO2恒温培养箱中培养1h,用完全培养基漂洗两遍后,在荧光显微镜下检测。根据QD试剂推荐浓度标记第3代细胞(P3)为试验组,以未用QD标记的细胞作为空白对照组。用苔盼蓝拒染法检测标记后细胞存活率,用MTT法观察染料对细胞增殖性的影响,采用茜素红、碱性磷酸酶染色、real-time PCR鉴定对成骨分化的影响；分别在标记后即刻、1w、2w、4w、6w利用荧光显微镜计数检测两组标记时间和阳性率；并用电镜观察标记后细胞在材料的贴附性。 3.华南理工大学材料学院提供胶原-生物活性玻璃,提取材料浸提液,实施溶血试验、细胞增殖试验,将细胞粘附材料分别于1d、3d、5d、7d进行对比电镜观察,并将SCs负载与胶原-生物活性玻璃上,采用电镜观察材料对SCs贴壁情况；采用胶原-生物活性玻璃在干湿态下,对湿态下胶原材料进行力学强度对比；取6块6x8mm2,采用微量注射器,每20gL液体量滴加材料,直至材料地面有液体渗出为止,统计注射液体量,计算最佳加液量；采用5点注射,分别于材料表面0mm、2mm、4mm进行梯度注射寻求最佳注射位点；取P3代BMSCs接种于材料及24孔板中,其中一部分材料采用SD大鼠骨髓间充质干细胞培养基培养,一部分材料采用成骨诱导,分别于与之相同培养基的细胞进行对比,2w后采用real-time PCR进行检测,检测成骨分化程度。取P3 BMSCs向成骨分化2周,种植在胶原-生物活性玻璃上,切成1ml薄片皮下包埋裸鼠背部,将观察材料有诱导成骨性能。 4.采用快速成型技术,自行设计塑料接骨板,材料为进口无毒材料。制备SD大鼠股骨8mm骨缺损。共采用12只350-500g大鼠处死,取出股骨进行接骨板固定与克氏针髓内固定,测试轴向生物力学强度,检测两种固定方式轴向抗压缩能力；取24只350-500g大鼠随机分为A组与B组,A组采用接骨板固定,B组采用克氏针固定。分别于术后2w、4w、8w进行X线片,评价固定缺损稳定状况,总结两组失败率及类型,总结适合大鼠最佳固定方式。 5.采用9只150-200g SD大鼠,暴露并游离隐动静脉隐神经束,移植于预先处理的胶原-生物活性玻璃,于术后3d、7d、14d采用冰冻切片进行HE染色观察血管网形成；采用GFP转染BMSCs向体外诱导成骨2周,采用Qtracker655标记大鼠SCs接种于胶原-生物活性玻璃上,移植于大鼠8mm骨缺损体内1周后取出采用共聚焦观察两种细胞示踪可行性。结果 1.原代SD大鼠BMSCs、OB、DRG、SCG、SCs培养成功,DRG于共培养5d时,对成骨细胞较空白对照组有明显的促增殖作用,有显著性差异(相应F=0.802,P=0.007)而与其他对照组及无明显差别,SCG各个时间段对颅骨来源成骨细胞较对照组均无促增殖作用(P0.05)。采用96孔共培养板,共培养1d、3d、5d无统计学差异,共培养7d、9d两个时间段SCs对成骨细胞有明显的促增殖作用,较对照组均有显著性差异(P0.05)。SCs对BMSCs来源OB在共培养1、3d两组没有统计学差异,余下3个时间段均有统计学差异,试验组由于对照组。SCs在3d、5d、7d对成骨细胞作用在ALP、OPN、OCN、BMP-2、Col1a mRNA较未共培养的成骨细胞均低表达,表明SCs对成骨细胞起抑制分化作用。而对BMSCs来源成骨细胞,在ALP、OPN、OCN、BMP-2、Col1a mRNA在3d是均有高表达,ALP、Col1a在第7d呈低表达,提示SCs在成骨诱导培养环境中对BMSCs来源的成骨细胞起到促分化作用。 2.试验组与对照组细胞存活率均90%,比较差异无统计学意义(P0.05)培养1、3、5、7、9 d两组增殖率比较差异均无统计学意义(P0.05)。标记的BMSCs经诱导分化2周后,茜素红及ALP染色均呈阳性,实时荧光定量PCR检测经标记的BMSCs的OPN mRNA、Bglap mRNA、Colla 1 mRNA、Alp1 mRNA、Bmp2 mRNA较对照组均呈高表达。荧光显微镜下胞浆呈红色荧光,标记后即可标记率可达96.5±1.59%,随着标记时间的增加,标记率下降,分别为1w 93.30±1.51%、2w 72.40±2.90%、4w 40.10±3.60%、6w10.00±1.70%,对照组各时间点标记阳性率均为0；扫描电镜观察标记后细胞和材料贴附良好。 3.采用胶原-生物活性玻璃材料中,采用支架材料浸提液,对兔静脉血没有产生溶血作用,对RSC96增殖较对照组在各个时间段均无抑制作用；采用轴向压缩测试力学强度,证实湿态下支架材料的强度高于胶原组织,而明显低于干性支架材料；采用逐渐加液体的方法,探讨出最佳加液量为0.88ml/cm3;采用五点梯度为2mm为最佳注射方法,可将细胞均匀种植到材料上；材料对BMSCs起到抑制分化作用；胶原-生物活性玻璃负载成骨细胞包埋裸鼠体内可以形成骨质,构建的组织工程骨具在BMP-2、OP、Col1a mRNA较正常骨高表达,提示组织工程骨处于快速成骨期。 4.克氏针髓内固定及接骨板固定两种方式手术技巧讨论,克氏针固定较接骨板固定更为方便,所需要时间更短,两者存在显著性差异(P0.05)。并术后2、4、8w进行X线片统计固定失效率,并在体外进行轴向压缩生物力学检测。探讨最佳固定方式。发现在体外生物力学强度测试中,接骨板轴向压缩所受力为40.38±4.04,克氏针压缩失效值所受力为29.53±2.95,克氏针彻底失效值所受力为58.49±5.85,三组间均有统计学差异(P0.05)。在动态X线片观察中发现,克氏针组出现失败率为91.67%(11／12),接骨板失败率为16.67%(2／12)。本部分研究得出在大鼠股骨骨缺损中,应采用接骨板固定更为理想。 5.在大鼠模型中可采用隐动脉、隐静脉、隐神经束作为整体血管神经束移植构建血管神经化组织工程骨,HE染色鉴定材料有较多血管形成,并随时间递增血管分布密度增加。可采用慢病毒GFP转染BMSCs以及QD655标记SCs种植到胶原-生物活性玻璃进行体内示踪。证实隐动脉、静脉、神经束可以作为整体进行移植,在材料内可以形成较多血管组织。在共聚焦显微镜下可以对两种细胞进行示踪,并在一周的X线片中可以发现有成骨形成。结论 1.DRG可能对成骨细胞起到促增殖作用；SCG对成骨细胞不起到促增殖作用；SCs对成骨细胞起到明显的促增殖作用；SCs对颅骨来源的成骨细胞起到抑制分化作用；对骨髓间充质干细胞来源的成骨细胞起到促进分化作用。 2. Qtraker655对大鼠BMSCs标记时间长,标记率及安全性高,并不改变细胞的成骨性能,是一种良好标记物。 3.胶原-生物活性玻璃是一种生物相容性好,可构建骨组织的支架材料。 4.在大鼠股骨骨缺损固定中,接骨板效果优于克氏针髓内固定。 5.在大鼠构建神经血管化组织工程,隐动静脉隐神经束可作为外源性因素移植到材料内进行构建。
[Abstract]:objective
1. primary cultured SD rat bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs), GFP transfected bone marrow mesenchymal stem cells (GFP-BMSCs), osteoblasts (osteoblasts, OB), Schwann cells (Schwann), sensory neurons, sympathetic neurons The effects of DRG, SCG and SCs on the proliferation of GFP-BMSCs differentiated osteoblasts were observed. The effect of.SCs on proliferation of BMSCs differentiated OB and OB derived from skull and its mechanism were studied.
2. study the feasibility of using fluorescent microsphere 655 (Quantum dot 655, QD655) to mark rat BMSCs in vitro, and explore the toxicity, proliferation, osteogenic differentiation and dynamic labeling rate of the dye to rat BMSCs, and lay a foundation for the study in vivo.
3. study the biocompatibility of collagen bioactive glass materials, including hemolysis test, RSC96 cell proliferation test, study on mechanical strength under dry and wet state of collagen bioactive glass, study on optimum inoculation liquid quantity, study on best inoculation mode, BMSCs and SCs adhesion to material proliferation study, and material to cell growth Colonization test and osteogenic differentiation test, and preliminary in vivo subcutaneous embedding verification osteogenesis test.
4. the study of the stability of 8mm bone defect in rats with Kirschner wire and rapid prototyping was studied. The axial compression biomechanics test and the dynamic observation of the fixed failure rate in the body were used to explore the methods of two fixed methods, surgical techniques, advantages and disadvantages.
5. the feasibility of synchronous construction of neurovascularized tissue engineering bone by using rat saphenous vein and saphenous nerve bundle as a neurovascular bundle as a exogenous factor, as well as the feasibility study of using fluorescent microspheres labeled SCs and GFP transfected osteoblasts combined with collagen bioactive glass in vivo.
Method
1. SD rats of 2 weeks old were used as experimental animal models. The cells were collected by femur and tibia bone marrow flushing method, and third generation of BMSCs were used for test. 2 rats of 1D aged milk rats were soaked with 75% alcohol and 15min was soaked in 75% alcohol. A sterile ophthalmic scissors were used to build 1-2mm3 fragments. 1mL0.25% trypsin 37 C was added to the broken bone tissue. 90min, slightly off, to the supernatant. Add 0.2% type of type I collagenase 3-5mL, 37 C, digest 2 times 60min. to stop digestion, collect cells, repeat the test, collect cells, grow in 25 cm2 culture bottle, put at 37, 5%CO2 incubator incubator.24h for liquid, and then every 2-3D change 1 times. To achieve 80%-90% fusion (primary culture 3-5d). The 2-5 generation of 15d pregnant rats were used for testing, using alkaline phosphatase staining to be used for identification, and used for test. Using 15d pregnant rats, sterile fetuses were taken out of fetal rats, the dorsal root ganglion was taken, 0.25% trypsin was digested 50min, and the sensory neurons were cultivated on the prepads, and the 1D old rats were used to take the superior cervical ganglion and the 0.25% pancreatin to digest 30min. To the pre plank culture plate, the 1D milk rat was taken out of the sciatic nerve and the brachial plexus, and the tissue block adherence was used to obtain the differentiation of SCs. with P3 BMSCs for 2 weeks, and the differentiation of osteoblasts was detected by SCs, DRG, SCG and calvarial osteoblasts and BMSCs derived osteoblasts, respectively, to detect the proliferation and differentiation of osteoblasts after co culture. Chemical mechanism.
2. using QD655 according to the recommended concentration of the kit, the QD reagent A and the reagent carrier B were mixed in the 1.5mL micro centrifuge tube, respectively, and placed in the incubating 15min, adding the 100gL medium, and adding the scroll 30s, adding 106 P3 cells to the 0.2mL suspense to the tube. After being beaten, it was placed at 37, and the 5%CO2 constant temperature incubator was cultivated for two times. After the fluorescence microscope, the third generation cells (P3) were labeled with the recommended concentration of QD as the experimental group, and the cells without QD were used as the blank control group. The cell viability was detected by the trypan blue staining method, and the effect of the dye on the cell proliferation was observed by MTT method. Alizarin red, alkaline phosphatase staining, and real-time PCR were used. The effect of 1W, 2W, 4W, and 6W on the two groups of labeling time and positive rate were detected by fluorescence microscopy, and the adherability of the labeled cells was observed by electron microscopy.
3. the College of materials of South China University of Technology provided collagen bioactive glass, extraction liquid extract, hemolysis test and cell proliferation test. Cell adhesion materials were observed by 1D, 3D, 5D, 7d respectively. The SCs load and collagen bioactive glass were used to observe the adherence of the materials to SCs by electron microscope, and the collagen bioactivity was used. The mechanical strength of the active glass was compared with the wet state of the collagen in wet state, and 6 pieces of 6x8mm2 were taken, using a microsyringe and adding materials per 20gL liquid, until the material ground had liquid exudation. The volume of the injection was calculated and the optimum amount of liquid was calculated; the gradient injection was carried out on the surface of the material, 0mm, 2mm, and 4mm respectively. The best injection site was sought, and P3 generation BMSCs was inoculated in material and 24 Kong Banzhong. Some of the materials were cultured in the medium of bone marrow mesenchymal stem cells in SD rats. A part of the material was induced by osteogenesis, and the cells were compared with the same medium. The degree of osteogenesis differentiation was detected by real-time PCR after 2W. P3 BMSCs was cultured on collagen bioactive glass for 2 weeks, and was cut into 1ml slices to subcutaneous nude mice.
4. using the rapid prototyping technology, the plastic bone plate was designed by ourselves, and the material was imported nontoxic material. The 8mm bone defect of SD rat was prepared. A total of 12 350-500g rats were killed, the femur was taken for the plate fixation and the Kirschner pin fixation, the axial biomechanical strength was tested, and the axial compression resistance of the two fixed methods was tested; 24 350 -500g rats were randomly divided into group A and group B, group A was fixed with osteosynthesis and group B was fixed with Kirschner pin. The X-ray films were performed on 2W, 4W, 8W after operation respectively. The stability of the fixed defect was evaluated, the failure rate and type of the two groups were summarized, and the best fixation method suitable for the rats was summed up.
5. 9 150-200g SD rats were used to expose and dissociate the saphenous saphenous nerve bundle of the saphenous vein, and transplanted into the pretreated collagen bioactive glass. After the operation, 3D, 7d, 14d were stained with frozen section to observe the formation of vascular network, and GFP transfected BMSCs to induce osteogenesis in vitro for 2 weeks, and the Qtracker655 labelled rat SCs was inoculated to the collagen biological activity. After 1 weeks of transplantation, the rats were transplanted into 8mm bone defect in rats. Confocal microscopy was used to observe the feasibility of two cell tracers.
Result
1. the primary SD rats BMSCs, OB, DRG, SCG, SCs were cultured successfully. When DRG was co cultured with 5D, the osteoblasts had obvious proliferation promoting effect compared with the blank control group. There was a significant difference (corresponding F=0.802, P=0.007) and no significant difference between the control group and the other control groups. All the periods of SCG on the skull had no proliferation effect on the skull derived osteoblasts compared with the control group (P0.05). 96 holes co culture plate, co culture 1D, 3D, 5D no statistical difference, co culture 7d, 9D two time period SCs to osteoblast proliferation effect, there is significant difference (P0.05).SCs to BMSCs source OB in the co culture 1,3d two groups of no statistical difference, the remaining 3 time periods have statistical differences, the test group because of the difference The effects of.SCs on osteoblasts in 3D, 5D, and 7d are low expressed in ALP, OPN, OCN, BMP-2, Col1a mRNA, indicating that SCs can inhibit the differentiation of osteoblasts. The culture environment promotes the differentiation of BMSCs derived osteoblasts.
The cell survival rate of 2. test group and control group was 90%, the difference was not statistically significant (P0.05), the proliferation rate of 1,3,5,7,9 D two groups was not statistically significant (P0.05). After 2 weeks of induced differentiation, alizarin red and ALP staining were positive, and real-time fluorescence PCR was used to detect OPN mRNA of labeled BMSCs, Bglap mRNA. 1 mRNA, Alp1 mRNA and Bmp2 mRNA were higher than the control group. The cytoplasm of the cytoplasm was red under the fluorescence microscope, and the labeling rate was 96.5 + 1.59%. With the increase of labeling time, the labeling rate decreased, respectively, 1W 93.30 + 1.51%, 2W 72.40 + 2.90%, 4W 40.10 + 3.60%, 6w10.00 + 1.70%, and the positive rate of each time point in the control group was 0. The cells and materials were well attached after scanning electron microscopy.
3. in collagen bioactive glass material, the use of scaffold material dipping solution did not produce hemolytic effect on rabbit venous blood. The proliferation of RSC96 was not inhibited in the control group at all time periods, and the mechanical strength of axial compression test proved that the strength of the scaffold material under wet state was higher than that of the collagen tissue, but it was obviously lower than that of the dry stents. Material; using the method of gradually adding liquid, the optimum adding amount is 0.88ml/cm3, and the five point gradient is 2mm as the best injection method, the cells can be planted evenly on the material; the material can inhibit the differentiation of BMSCs; the collagen bioactive glass loaded osteoblast can form the bone in the nude mice and construct the tissue worker. The BMP-2, OP and Col1a mRNA were higher than normal bone, suggesting that the tissue-engineered bone was in a fast osteogenic stage.
4. Kirschner pin intramedullary fixation and bone plate fixation two methods of surgical discussion, Kirschner pin fixation more convenient than the plate fixation, the need for shorter time, there is a significant difference between the two (P0.05). And postoperative 2,4,8w X ray statistical fixed failure rate, and in vitro axial compression biomechanical test. To explore the best fixation method. It was found that in the biomechanical strength test in vitro, the force of the axial compression of the plate was 40.38 + 4.04, the force of the Kirschner's needle compression failure was 29.53 + 2.95, the force of the Kirschner's total invalidation value was 58.49 + 5.85. There were statistical differences between the three groups (P0.05). The failure rate of the Kirschner needle group was 91.67% (11 / 12) in the dynamic X-ray observation. The failure rate of the plate was 16.67% (2 / 12). In this part of the study, it was suggested that the fixation of the bone plate should be more ideal in the femoral bone defect of rats.
5. in rat model, saphenous artery, saphenous vein and saphenous nerve bundle can be used as a whole vascular nerve bundle to construct vascular neurovascular tissue engineering bone. HE staining is used to identify more blood vessels and increase the density of vascular distribution with time. Lentivirus GFP transfection BMSCs and QD655 labeled SCs can be used to grow collagen bioactive glass. The body is traced in the body. It is confirmed that the saphenous artery, vein, and nerve bundle can be transplanted as a whole, and more vascular tissue can be formed in the material. Two cells can be traced under confocal microscopy, and the formation of osteogenesis can be found in a week's X-ray film.
conclusion
1.DRG may promote osteoblast proliferation; SCG does not promote osteoblast proliferation; SCs plays a significant role in promoting osteoblast proliferation; SCs inhibits the differentiation of osteoblasts derived from the skull and promotes differentiation of osteoblasts derived from bone marrow mesenchymal stem cells.
2. Qtraker655 is a good marker for BMSCs long labeling time, high labeling rate and high safety.
3. collagen bioactive glass is a scaffold material with good biocompatibility and can be used to construct bone tissue.
4. in the fixation of femoral bone defects in rats, the effect of the plate is better than that of the Kirschner wire.
5. in the construction of neurovascular tissue engineering in rats, saphenous saphenous nerve bundles can be transplanted into the materials as exogenous factors.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【参考文献】