本文選題：骨組織工程 + 增殖　； 參考：《南方醫(yī)科大學(xué)》2010年博士論文

【摘要】：目的 1.體外原代培養(yǎng)SD大鼠骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cells, BMSCs)、GFP轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞(GFP-BMSCs)、成骨細(xì)胞(osteoblasts, OB)、雪旺細(xì)胞(Schwann cells,SCs)、感覺神經(jīng)元(dorsal root ganglion,DRG)、交感神經(jīng)元(superior cervical ganglion, SCG)細(xì)胞。觀察DRG、SCG、SCs對(duì)GFP-BMSCs分化的成骨細(xì)胞增殖的影響。SCs對(duì)BMSCs分化OB及顱骨來源OB增殖的影響及機(jī)制研究。 2.研究采用熒光微球655 (Quantum dot 655, QD655)標(biāo)記大鼠BMSCs體外可行性研究。探討此染料對(duì)大鼠BMSCs的毒性、增殖、成骨分化、動(dòng)態(tài)標(biāo)記率等功能,為體內(nèi)示蹤研究打下研究基礎(chǔ)。 3.研究膠原-生物活性玻璃材料的生物相容性研究,包括溶血試驗(yàn),浸提液對(duì)RSC96細(xì)胞株增殖試驗(yàn),膠原-生物活性玻璃干濕態(tài)下力學(xué)強(qiáng)度研究,最佳接種液體量研究,最佳接種方式方式研究、BMSCs與SCs粘附于材料增殖研究,材料對(duì)細(xì)胞促增殖試驗(yàn)及向成骨促分化試驗(yàn),以及初步體內(nèi)皮下包埋驗(yàn)證成骨試驗(yàn)。 4.探討采用克氏針與快速成型技術(shù)制成接骨板固定大鼠8mm骨缺損穩(wěn)定性研究,采用體內(nèi)軸向壓縮生物力學(xué)測(cè)試及體內(nèi)動(dòng)態(tài)觀察固定失效率進(jìn)行試驗(yàn),探求兩種固定方式的方法、手術(shù)技巧、優(yōu)點(diǎn)及缺點(diǎn),探求合適固定大鼠骨缺損固定方式。 5.探討采用大鼠隱動(dòng)靜脈及隱神經(jīng)束作為神經(jīng)血管束移植作為外源性因素同步構(gòu)建神經(jīng)血管化組織工程骨的可行性,以及采用熒光微球標(biāo)記的SCs與GFP轉(zhuǎn)染的成骨細(xì)胞聯(lián)合負(fù)載于膠原-生物活性玻璃體內(nèi)示蹤可行性研究。 方法 1.以2周齡SD大鼠為試驗(yàn)動(dòng)物模型,采用股骨、脛骨骨髓沖洗法收集細(xì)胞,以培養(yǎng)的第3代BMSCs用于試驗(yàn)；采用1d齡乳鼠2只,采用75%酒精浸泡15min,無菌取出顱骨,采用無菌眼科剪建成1-2mm3碎片。向剪碎的骨組織中加入1mL0.25%的胰蛋白酶37℃,消化90min,稍離,去上清。加入0.2%Ⅰ型膠原酶3-5mL,37℃,連續(xù)消化2次60min。中止消化,收集細(xì)胞,重復(fù)上述試驗(yàn),收集細(xì)胞,計(jì)數(shù)后,種植于25 cm2培養(yǎng)瓶中,置于37℃、5%CO2恒溫培養(yǎng)箱中培養(yǎng)。24h后換液,以后每2-3d換液1次。達(dá)到80%-90%融合時(shí)(原代培養(yǎng)3-5d),消化傳代,分組培養(yǎng),采用堿性磷酸酶染色進(jìn)行鑒定,2-5代用于試驗(yàn)；采用15d孕鼠,無菌取出胎鼠,取背根神經(jīng)節(jié),0.25%胰酶消化50min,種植到預(yù)先鋪板的蓋玻片上,進(jìn)行感覺神經(jīng)元培養(yǎng)；采用1d齡乳鼠,取頸上神經(jīng)節(jié),0.25%胰酶消化30min,種植到預(yù)先鋪板的培養(yǎng)板；取1d乳鼠,取出坐骨神經(jīng)及臂叢神經(jīng),采用組織塊貼壁法獲得SCs。采用P3 BMSCs向成骨誘導(dǎo)分化2周,進(jìn)行鑒定；分別采用SCs、DRG、SCG與顱骨來源成骨細(xì)胞和BMSCs來源成骨細(xì)胞共培養(yǎng)檢測(cè),檢測(cè)共培養(yǎng)后成骨細(xì)胞增殖及分化機(jī)制。 2.采用QD655按照試劑盒推薦濃度,分別將QD試劑A和試劑載體B各取1μL混置于1.5mL微量離心管中,放于孵育15min,加入100gL培養(yǎng)基,渦旋30s,向管中加入含106個(gè)P3細(xì)胞的0.2mL懸液,打勻后置于37℃、5%CO2恒溫培養(yǎng)箱中培養(yǎng)1h,用完全培養(yǎng)基漂洗兩遍后,在熒光顯微鏡下檢測(cè)。根據(jù)QD試劑推薦濃度標(biāo)記第3代細(xì)胞(P3)為試驗(yàn)組,以未用QD標(biāo)記的細(xì)胞作為空白對(duì)照組。用苔盼藍(lán)拒染法檢測(cè)標(biāo)記后細(xì)胞存活率,用MTT法觀察染料對(duì)細(xì)胞增殖性的影響,采用茜素紅、堿性磷酸酶染色、real-time PCR鑒定對(duì)成骨分化的影響；分別在標(biāo)記后即刻、1w、2w、4w、6w利用熒光顯微鏡計(jì)數(shù)檢測(cè)兩組標(biāo)記時(shí)間和陽性率；并用電鏡觀察標(biāo)記后細(xì)胞在材料的貼附性。 3.華南理工大學(xué)材料學(xué)院提供膠原-生物活性玻璃,提取材料浸提液,實(shí)施溶血試驗(yàn)、細(xì)胞增殖試驗(yàn),將細(xì)胞粘附材料分別于1d、3d、5d、7d進(jìn)行對(duì)比電鏡觀察,并將SCs負(fù)載與膠原-生物活性玻璃上,采用電鏡觀察材料對(duì)SCs貼壁情況；采用膠原-生物活性玻璃在干濕態(tài)下,對(duì)濕態(tài)下膠原材料進(jìn)行力學(xué)強(qiáng)度對(duì)比；取6塊6x8mm2,采用微量注射器,每20gL液體量滴加材料,直至材料地面有液體滲出為止,統(tǒng)計(jì)注射液體量,計(jì)算最佳加液量；采用5點(diǎn)注射,分別于材料表面0mm、2mm、4mm進(jìn)行梯度注射尋求最佳注射位點(diǎn)；取P3代BMSCs接種于材料及24孔板中,其中一部分材料采用SD大鼠骨髓間充質(zhì)干細(xì)胞培養(yǎng)基培養(yǎng),一部分材料采用成骨誘導(dǎo),分別于與之相同培養(yǎng)基的細(xì)胞進(jìn)行對(duì)比,2w后采用real-time PCR進(jìn)行檢測(cè),檢測(cè)成骨分化程度。取P3 BMSCs向成骨分化2周,種植在膠原-生物活性玻璃上,切成1ml薄片皮下包埋裸鼠背部,將觀察材料有誘導(dǎo)成骨性能。 4.采用快速成型技術(shù),自行設(shè)計(jì)塑料接骨板,材料為進(jìn)口無毒材料。制備SD大鼠股骨8mm骨缺損。共采用12只350-500g大鼠處死,取出股骨進(jìn)行接骨板固定與克氏針?biāo)鑳?nèi)固定,測(cè)試軸向生物力學(xué)強(qiáng)度,檢測(cè)兩種固定方式軸向抗壓縮能力；取24只350-500g大鼠隨機(jī)分為A組與B組,A組采用接骨板固定,B組采用克氏針固定。分別于術(shù)后2w、4w、8w進(jìn)行X線片,評(píng)價(jià)固定缺損穩(wěn)定狀況,總結(jié)兩組失敗率及類型,總結(jié)適合大鼠最佳固定方式。 5.采用9只150-200g SD大鼠,暴露并游離隱動(dòng)靜脈隱神經(jīng)束,移植于預(yù)先處理的膠原-生物活性玻璃,于術(shù)后3d、7d、14d采用冰凍切片進(jìn)行HE染色觀察血管網(wǎng)形成；采用GFP轉(zhuǎn)染BMSCs向體外誘導(dǎo)成骨2周,采用Qtracker655標(biāo)記大鼠SCs接種于膠原-生物活性玻璃上,移植于大鼠8mm骨缺損體內(nèi)1周后取出采用共聚焦觀察兩種細(xì)胞示蹤可行性。 結(jié)果 1.原代SD大鼠BMSCs、OB、DRG、SCG、SCs培養(yǎng)成功,DRG于共培養(yǎng)5d時(shí),對(duì)成骨細(xì)胞較空白對(duì)照組有明顯的促增殖作用,有顯著性差異(相應(yīng)F=0.802,P=0.007)而與其他對(duì)照組及無明顯差別,SCG各個(gè)時(shí)間段對(duì)顱骨來源成骨細(xì)胞較對(duì)照組均無促增殖作用(P0.05)。采用96孔共培養(yǎng)板,共培養(yǎng)1d、3d、5d無統(tǒng)計(jì)學(xué)差異,共培養(yǎng)7d、9d兩個(gè)時(shí)間段SCs對(duì)成骨細(xì)胞有明顯的促增殖作用,較對(duì)照組均有顯著性差異(P0.05)。SCs對(duì)BMSCs來源OB在共培養(yǎng)1、3d兩組沒有統(tǒng)計(jì)學(xué)差異,余下3個(gè)時(shí)間段均有統(tǒng)計(jì)學(xué)差異,試驗(yàn)組由于對(duì)照組。SCs在3d、5d、7d對(duì)成骨細(xì)胞作用在ALP、OPN、OCN、BMP-2、Col1a mRNA較未共培養(yǎng)的成骨細(xì)胞均低表達(dá),表明SCs對(duì)成骨細(xì)胞起抑制分化作用。而對(duì)BMSCs來源成骨細(xì)胞,在ALP、OPN、OCN、BMP-2、Col1a mRNA在3d是均有高表達(dá),ALP、Col1a在第7d呈低表達(dá),提示SCs在成骨誘導(dǎo)培養(yǎng)環(huán)境中對(duì)BMSCs來源的成骨細(xì)胞起到促分化作用。 2.試驗(yàn)組與對(duì)照組細(xì)胞存活率均90%,比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)培養(yǎng)1、3、5、7、9 d兩組增殖率比較差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。標(biāo)記的BMSCs經(jīng)誘導(dǎo)分化2周后,茜素紅及ALP染色均呈陽性,實(shí)時(shí)熒光定量PCR檢測(cè)經(jīng)標(biāo)記的BMSCs的OPN mRNA、Bglap mRNA、Colla 1 mRNA、Alp1 mRNA、Bmp2 mRNA較對(duì)照組均呈高表達(dá)。熒光顯微鏡下胞漿呈紅色熒光,標(biāo)記后即可標(biāo)記率可達(dá)96.5±1.59%,隨著標(biāo)記時(shí)間的增加,標(biāo)記率下降,分別為1w 93.30±1.51%、2w 72.40±2.90%、4w 40.10±3.60%、6w10.00±1.70%,對(duì)照組各時(shí)間點(diǎn)標(biāo)記陽性率均為0；掃描電鏡觀察標(biāo)記后細(xì)胞和材料貼附良好。 3.采用膠原-生物活性玻璃材料中,采用支架材料浸提液,對(duì)兔靜脈血沒有產(chǎn)生溶血作用,對(duì)RSC96增殖較對(duì)照組在各個(gè)時(shí)間段均無抑制作用；采用軸向壓縮測(cè)試力學(xué)強(qiáng)度,證實(shí)濕態(tài)下支架材料的強(qiáng)度高于膠原組織,而明顯低于干性支架材料；采用逐漸加液體的方法,探討出最佳加液量為0.88ml/cm3;采用五點(diǎn)梯度為2mm為最佳注射方法,可將細(xì)胞均勻種植到材料上；材料對(duì)BMSCs起到抑制分化作用；膠原-生物活性玻璃負(fù)載成骨細(xì)胞包埋裸鼠體內(nèi)可以形成骨質(zhì),構(gòu)建的組織工程骨具在BMP-2、OP、Col1a mRNA較正常骨高表達(dá),提示組織工程骨處于快速成骨期。 4.克氏針?biāo)鑳?nèi)固定及接骨板固定兩種方式手術(shù)技巧討論,克氏針固定較接骨板固定更為方便,所需要時(shí)間更短,兩者存在顯著性差異(P0.05)。并術(shù)后2、4、8w進(jìn)行X線片統(tǒng)計(jì)固定失效率,并在體外進(jìn)行軸向壓縮生物力學(xué)檢測(cè)。探討最佳固定方式。發(fā)現(xiàn)在體外生物力學(xué)強(qiáng)度測(cè)試中,接骨板軸向壓縮所受力為40.38±4.04,克氏針壓縮失效值所受力為29.53±2.95,克氏針徹底失效值所受力為58.49±5.85,三組間均有統(tǒng)計(jì)學(xué)差異(P0.05)。在動(dòng)態(tài)X線片觀察中發(fā)現(xiàn),克氏針組出現(xiàn)失敗率為91.67%(11／12),接骨板失敗率為16.67%(2／12)。本部分研究得出在大鼠股骨骨缺損中,應(yīng)采用接骨板固定更為理想。 5.在大鼠模型中可采用隱動(dòng)脈、隱靜脈、隱神經(jīng)束作為整體血管神經(jīng)束移植構(gòu)建血管神經(jīng)化組織工程骨,HE染色鑒定材料有較多血管形成,并隨時(shí)間遞增血管分布密度增加�？刹捎寐《綠FP轉(zhuǎn)染BMSCs以及QD655標(biāo)記SCs種植到膠原-生物活性玻璃進(jìn)行體內(nèi)示蹤。證實(shí)隱動(dòng)脈、靜脈、神經(jīng)束可以作為整體進(jìn)行移植,在材料內(nèi)可以形成較多血管組織。在共聚焦顯微鏡下可以對(duì)兩種細(xì)胞進(jìn)行示蹤,并在一周的X線片中可以發(fā)現(xiàn)有成骨形成。 結(jié)論 1.DRG可能對(duì)成骨細(xì)胞起到促增殖作用；SCG對(duì)成骨細(xì)胞不起到促增殖作用；SCs對(duì)成骨細(xì)胞起到明顯的促增殖作用；SCs對(duì)顱骨來源的成骨細(xì)胞起到抑制分化作用；對(duì)骨髓間充質(zhì)干細(xì)胞來源的成骨細(xì)胞起到促進(jìn)分化作用。 2. Qtraker655對(duì)大鼠BMSCs標(biāo)記時(shí)間長,標(biāo)記率及安全性高,并不改變細(xì)胞的成骨性能,是一種良好標(biāo)記物。 3.膠原-生物活性玻璃是一種生物相容性好,可構(gòu)建骨組織的支架材料。 4.在大鼠股骨骨缺損固定中,接骨板效果優(yōu)于克氏針?biāo)鑳?nèi)固定。 5.在大鼠構(gòu)建神經(jīng)血管化組織工程,隱動(dòng)靜脈隱神經(jīng)束可作為外源性因素移植到材料內(nèi)進(jìn)行構(gòu)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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