本文選題：羊　切入點：同種異體　出處：《中國人民解放軍醫(yī)學院》2014年博士論文　論文類型：學位論文

【摘要】：第一部羊松質(zhì)凍干骨的制備及生物學檢測 目的：制備羊松質(zhì)凍干骨（FDB），研究其大體微觀結構特點，檢測生物力學參數(shù)，為羊松質(zhì)凍干骨的應用研究提供理論數(shù)據(jù)。 方法：通過脫脂脫蛋白及冷凍輻照等方法制備羊松質(zhì)凍干骨和未凍干的深凍骨。觀察大體形態(tài)和結構；掃描電子顯微鏡（SEM）觀察其超微結構，測量孔隙大��；微-CT測量計算其骨形態(tài)計量學參數(shù)； MTS生物力學機進行力學性能檢測，隨機附帶軟件記錄其最大載荷和應力-應變曲線，計算力學參數(shù)。 結果：制備成大小為10×10×10mm3羊髂骨松質(zhì)凍干骨塊及深凍骨塊。大體觀察見羊松質(zhì)凍干骨塊外觀呈乳白色，透亮，掃描電子顯微鏡下羊松質(zhì)凍干骨塊呈三維多孔網(wǎng)狀結構，孔隙相互連通，測量孔徑大小為268.60±40.01um。微-CT形態(tài)計量分析顯示羊松質(zhì)凍干骨的形態(tài)計量參數(shù)，BMD是317.98±46.70mg/cc，TMD是465.41±42.83mg/cc，BVF是0.4861±0.1135，Tb.Th.是0.1601±0.0340mm， Tb.SP.是0.1729±0.0506mm， Tb.n.是3.0322±0.3081/mm。力學測試結果：凍干骨與深凍骨的最大抗壓力分別是663.57±20.98Nu和430.41±31.47Nu；最大壓縮強度分別為8.19±0.80MPa和5.31±0.62MPa；彈性模量分別為289.45±30.10MPa和23.37±3.45MPa。 結論：羊松質(zhì)凍干骨具有作為支架材料的理想孔隙大小及孔隙率，有一定的生物力學抗壓性能，是一種理想的骨組織工程支架材料。 第二部分羊松質(zhì)凍干骨復合間充質(zhì)干細胞的體外實驗研究 目的：制備骨髓間充質(zhì)干細胞（BMSCs），研究其生物學特性、向成骨細胞分化的能力，及與同種異體松質(zhì)凍干骨復合的生物相容性。 方法：聯(lián)合密度梯度離心法和貼壁法對BMSCs進行分離及傳代培養(yǎng)。取第2、5、8代的BMSCs培養(yǎng)觀察并繪制細胞生長曲線；使用成骨誘導液對BMSCs進行成骨誘導，用茜素紅染色和堿性磷酸酶染色檢測BMSCs的成骨性能。將第3代BMSCs與羊松質(zhì)凍干骨塊在體外復合培養(yǎng)。通過掃描電鏡觀察細胞在松質(zhì)凍干骨上的附著生長情況。 結果：間充質(zhì)干細胞分離24h后，，部分細胞貼壁，多以圓形、橢圓形等不規(guī)則形態(tài)散在分布，4d后細胞呈典型的成纖維狀，開始形成小的集落，6d后細胞集落增多，并逐漸連成片狀。10d后各細胞集落之間逐漸相連，相互融合，鋪滿整個瓶底。傳代培養(yǎng)的干細胞懸于培養(yǎng)液中，2h散開貼壁，第2d全部貼壁，3-5d時達90%。BMSCs的生長曲線呈S型，P2、P5、P8細胞的生長曲線形態(tài)基本一致，第1-2d為潛伏期，生長較慢；3d后細胞增長速度加快，進入對數(shù)期；第7-8d停止生長，進入平臺期。茜素紅染色檢測誘導細胞的分化情況，在12-14d時出現(xiàn)分化跡象，有點狀的鈣化斑，且分布均勻，16d左右有礦化骨結節(jié)出現(xiàn)，第21d時骨結節(jié)形成比較明顯。堿性磷酸酶染色結果顯示BMSCs經(jīng)定向誘導21d后出現(xiàn)小的點狀鈣化斑。BMSCs和羊松質(zhì)凍干骨的復合物（MSCs-FDB）上有細胞生長。2h可見細胞貼附在FDB孔隙內(nèi)部及表面，胞體較小，細胞與骨小梁結合不太緊密，隨著培養(yǎng)時間增加，24h時胞體增大，生出突觸，與材料結合緊密。48h時，細胞呈不規(guī)則形狀，部分細胞形成小的集落，細胞功能活躍，胞體扁平，貼附在小梁骨表面。 結論：BMSCs具有自我增殖與自我更新能力，P2、P5、P8的細胞均有很良的生長性能，P3在成骨誘導劑作用下向成骨細胞定向分化，與同種異體松質(zhì)凍干骨組織相容性好。 第三部分羊MSCs-CFDB復合體成骨行為的體內(nèi)研究 目的：研究MSCs-CFDB復合體在大動物體內(nèi)的成骨行為，為同種異體MSCs-CFDB復合體在臨床使用提供實驗依據(jù)。 方法：將9只2.5-3年齡40-50kg的實驗綿羊，速眠新Ⅱ注射液麻醉后，在羊脛骨內(nèi)側(cè)距膝關節(jié)面下3-5mm處，截骨形成10×10×10mm3干骺端骨缺損模型。A組(實驗組)：將培養(yǎng)24h的MSCs-FDB復合體填充左下肢骨缺損區(qū)。對照組用DMEM液培養(yǎng)24h的羊松質(zhì)凍干骨植入右下肢相應部位骨缺損區(qū)。術后觀察實驗動物的一般活動情況及傷口愈合情況；分別術后4w、8w、12w隨機各處死3只動物，取植骨塊表面軟組織和3cm×3cm×3cm大小骨標本，微-CT檢查并重建骨小梁形態(tài)結構，分析測量主要骨形態(tài)計量參數(shù)包括骨礦物質(zhì)密度(BMD)、組織骨密度（TMD）、骨體積分數(shù)（BVF）、骨小梁厚度(Tb.Th.)、骨小梁間隙寬度(Tb.Sp.)和骨小梁數(shù)量(Tb.n.)。脫鈣后行HE染色和Masson三色染色，分析植入物的成骨情況。 結果：術后實驗動物活動正常，切口正常愈合。大體觀察植骨周圍軟組織，術后4w見表面纖維組織疏松，淡粉色，于8w、12w時，逐漸變致密，顏色漸接近周圍正常骨膜組織，無感染；組織切片未見淋巴浸潤。微-CT顯示實驗組與對照組骨組織結構變化趨勢一致，術后4w時骨小梁結構略增粗，孔隙增大，8w時小梁結構增粗，12w時骨小梁結構再次變細，孔隙增多變小，于所在部位的骨結構整合，不能區(qū)分。微-CT檢測移植物在4w、8w和12w時的骨礦物質(zhì)密度BMD：實驗組為145.10±19.96mg/cc，238.41±33.73mg/cc和215.30±28.50mg/cc；對照組為134.08±23.79mg/cc，240.20±23.02mg/cc和218.70±31.60mg/cc。實驗組的BVF在4w、8w和12w時分別為0.3772±0.0474，0.6038±0.1099和0.4016±0.0575；對照組為0.3629±0.0510，0.5967±0.0699和0.4453±0.0503；實驗組和對照組4w、8w、12w時的Tb.Th.分別為0.1535±0.0282mm和0.1600±0.0258mm，0.2974±0.0448mm和0.2830±0.0445mm，0.1710±0.0239mm和0.1650±0.0224mm；實驗組和對照組的Tb.Sp.在4w、8w、12w時分別為0.2591±0.0360mm和0.2932±0.0359mm，0.1866±0.0254mm和0.2193±0.0382mm，0.2681±0.0342mm和0.2273±0.0332mm。實驗組和對照組的Tb.n.在4w、8w、12w時分別為2.5573±0.3052mm和2.2531±0.3610mm，2.2149±0.2599mm和2.2469±0.2650mm，2.4632±0.2844mm和2.8050±0.3384mm。組織學檢查顯示實驗組與對照組骨組織結構變化趨勢基本一致，術后4w時骨小梁結構稀疏，略增粗，孔隙增大，有新纖維骨生成，在原骨小梁的周邊可見條狀編織骨，原骨小梁組織少部降解,無炎癥細胞；8w時骨小梁結構增粗，小梁變密，新骨的形成及移植骨的降解都明顯增加，出現(xiàn)新骨舊骨的相互交織狀態(tài)；12w時骨小梁結構變細，孔隙增多變小，原骨小梁組織大部分被取代，新骨逐漸向成熟骨組織轉(zhuǎn)變�？傮w上，MSCs-FDB組的變化趨勢比單純FDB組更快更明顯。Mason三色染色于術后4w可見兩組中紅色骨纖維周邊少量藍綠色骨纖維，8w時藍綠色新生骨纖維組織增多，與紅色纖維組織骨相互交織包繞，組織內(nèi)細胞核明顯。12w時可見大量藍綠色新生骨組織，有少量紅色纖維組織，重建的骨小梁規(guī)整。與實驗組相比，這一變化過程在對照組中發(fā)生緩慢。 結論：羊松質(zhì)凍干骨復合骨髓間充質(zhì)干細胞后在同種異體干骺端有較好的成骨性能。但在成骨過程的4w時，骨骼總量明顯下降，影響其生物學性能的組織結構發(fā)生變化。
[Abstract]:Preparation and biological detection of the first lyophilized bone of sheep pine
Objective: to prepare sheep pine cancellous bone (FDB), study its general microstructure characteristics, detect biomechanical parameters, and provide theoretical data for the application research of sheep pine lyophilized bone.
Methods: through degreasing deproteinization and freeze irradiation preparation method of sheep cancellous freeze-dried bone and freeze-dried freeze-dried bone. To observe the general morphology and structure; scanning electron microscope (SEM) to observe the ultrastructure, measuring the pore size; micro -CT to measure the parameters of bone histomorphometry; MTS biomechanical machine the mechanical properties testing, random with software to record the maximum load and stress-strain curve, calculation of mechanical parameters.
Results: the preparation of size 10 * 10 * 10mm3 sheep iliac cancellous bone and freeze-dried bone deep freeze. There was no sheep cancellous and freeze-dried bone block appearance is milky white, translucent, scanning electron microscope and freeze-dried bone sheep cancellous block is a three-dimensional porous structure, pore connectivity. Measuring the size of the hole for the analysis of 268.60 + 40.01um. micro -CT display morphometry morphometric parameters of sheep cancellous freeze-dried bone, BMD is 317.98 + 46.70mg/cc, TMD + 42.83mg/cc is 465.41, BVF is 0.4861 + 0.1135, Tb.Th. is 0.1601 + 0.0340mm, Tb.SP. + 0.0506mm is 0.1729, Tb.n. is 3.0322 + 0.3081/mm.: mechanical test results the freeze-dried bone and the maximum anti pressure of deep frozen bone were 663.57 + 20.98Nu and 430.41 + 31.47Nu; the maximum compressive strength were 8.19 + 0.80MPa and 5.31 + 0.62MPa; elastic modulus were 289.45 + 30.10MPa and 23.37 + 3.45MPa.
Conclusion: sheep cancellous freeze-dried bone has ideal pore size and porosity as scaffold material, and has certain biomechanical and compressive properties. It is an ideal scaffold material for bone tissue engineering.
In vitro experimental study on the second part of lamb cancellous freeze-dried bone mesenchymal stem cells in vitro
Objective: to prepare bone marrow mesenchymal stem cells (BMSCs) and study their biological characteristics, their ability to differentiate into osteoblasts, and their biocompatibility with the allogeneic cancellous bone.
Methods: the combination of density gradient centrifugation and adherent method were separated and cultured on BMSCs. The 2,5,8 generation of BMSCs culture observation and cell growth curve; using the osteoblast of BMSCs osteogenic induction medium, osteogenic properties of BMSCs was detected by alizarin red staining and alkaline phosphatase. The third generation of BMSCs and the sheep cancellous bone in the freeze-dried cultured. Cells were observed by scanning electron microscope in the freeze-dried cancellous bone. The attached growth situation
Results: mesenchymal stem cells isolated from 24h, some adherent cells, mostly round, oval and irregular scattered 4D cells were fibrous into typical, began to form small colonies, 6D cell colonies increased, and gradually into a slice.10d after each cell colony between gradually connected to each other, covered the entire bottom of the bottle. The cultured stem cells suspended in the culture medium, 2h dispersed adherent 2D all adherent growth curve 3-5d of 90%.BMSCs was S, P2, P5, the growth curve of P8 cells are basically the same, the 1-2D is the incubation period of slower growth 3D; cell growth speed, the logarithmic phase; the 7-8d stopped growing into the platform. The differentiation of alizarin red staining cells, showing signs of differentiation in 12-14d, with punctate calcification, and uniform distribution, about 16d with mineralized bone nodule, 21d bone nodule formation Become more obvious. Alkaline phosphatase staining showed that BMSCs complex by directional after 21d induced by small punctate calcified plaque in.BMSCs and lyophilized sheep cancellous bone (MSCs-FDB) on cell growth.2h cells were attached to FDB internal pore and surface, smaller cell body, cells and bone trabeculae with not too close and as the culture time increased, body size, 24h cells produce.48h synapses, closely combined with material, cells with irregular shape, part of the cells formed small colonies, cell function active, cell body flat, attached to the trabecular bone surface.
Conclusion: BMSCs has the ability of self proliferation and self-renewal. The cells of P2, P5 and P8 have good growth performance. P3 can differentiate into osteoblasts under the action of osteogenic inducers, and has good histocompatibility with lyophilized allogenic cancellous bone.
In vivo study of the osteogenesis of the third part of the sheep MSCs-CFDB complex
Objective: To study the osteogenic behavior of MSCs-CFDB complex in large animals, and to provide experimental basis for the clinical use of allogenic MSCs-CFDB complex.
Methods: the 9 sheep 2.5-3 age 40-50kg, anesthesia of Sumianxin II after injection in sheep from the medial tibial articular surface under 3-5mm, bone formation of 10 x 10 x 10mm3 metaphyseal bone defect model group.A (experimental group): the culture complex of MSCs-FDB 24h filling the left lower limb bone defect the control group using DMEM. Culture fluid 24h sheep cancellous bone implant freeze-dried right lower limb bone defect area. The corresponding parts of general activity and observation of postoperative wound healing of experimental animal; respectively after 4W, 8W, 12W were randomly sacrificed 3 animal bone grafting surface of soft tissue and 3cm * the size of 3cm * 3cm bone specimens, micro -CT check and build the trabecular bone structure, bone histomorphometry analysis mainly includes measuring bone mineral density (BMD), bone density (TMD), bone volume fraction (BVF), trabecular thickness (Tb.Th.), trabecular gap width (Tb.Sp.) and trabecular bone volume (Tb.n. After decalcification, HE staining and Masson trichromatic staining were used to analyze the osteogenesis of the implant.
Results: after experiment animal activity is normal, normal incision healing. General observation of bone surrounding soft tissue, postoperative 4W superficial fibrous tissue loose, pale pink, 8W, 12W, became denser, the color gradually close to the surrounding normal periosteum, no infection; invasive tissue sections showed no lymph micro -CT display experiment. Group and control group consistent trend of structure of bone tissue, postoperative 4W trabecular structure slightly coarse, pore diameter and 8W trabecular thickening, 12W trabecular structure again became thin, pores become smaller, bone structure integration, on location cannot be distinguished. The micro detection of -CT graft in 4W, BMD, 8W and 12W in bone mineral density in the experimental group was 145.10 + 19.96mg/cc, 238.41 + 33.73mg/cc and 215.30 + 28.50mg/cc; the control group was 134.08 + 23.79mg/cc, 240.20 + 23.02mg/cc and 218.70 + 31.60mg/cc. group BVF in 4W, 8W and 12W respectively 0.3772. 0.0474,0.6038 + 0.1099 and 0.4016 + 0.0575; the control group was 0.3629 + 0.0510,0.5967 + 0.0699 and 0.4453 + 0.0503; the experimental group and the control group 4W, 8W, 12W and Tb.Th. were respectively 0.1535 + 0.0282mm and 0.1600 + 0.0258mm, 0.2974 + 0.0448mm and 0.2830 + 0.0445mm, 0.1710 + 0.0239mm and 0.1650 + 0.0224mm; the experimental group and the control group Tb.Sp. in 4W, 8W, 12W were 0.2591 + 0.0360mm and 0.2932 + 0.0359mm, 0.1866 + 0.0254mm and 0.2193 + 0.0382mm, 0.2681 + 0.0342mm and 0.2273 + 0.0332mm. experimental group and control group Tb.n. in 4W, 8W, 12W were 2.5573 + 0.3052mm and 2.2531 + 0.3610mm, 2.2149 + 0.2599mm and 2.2469 + 0.2650mm, 2.4632 + 0.2844mm and 2.8050 + 0.3384mm. histological examination showed that the experimental group was consistent with the change in the control group bone tissue structure trend, 4W after operation of bone trabecular structure sparse, slightly coarse, pore diameter and new bone formation in the fiber. The original strip seen around the trabeculae of woven bone, bone trabecular tissue of degradation, no inflammatory cells; 8W trabecular structure thickening, trabecular bone formation and Bianmi degradation, new bone were significantly increased, the emergence of new bone intertwined state old bone 12W bone; Liang Jie the thinner, smaller pores, the original trabecular bone most replaced by new bone gradually transformed into mature bone tissue. On the whole, the change trend of the MSCs-FDB group than the FDB group more obvious.Mason trichrome staining after 4W visible red bone fiber in two groups around the small blue green color fiber bone 8W, when the blue and green new bone fibrous tissue increased, interwoven with red bone fibrous tissue wrapping, tissue.12w nuclei obviously shows a large number of blue and green new bone tissue, a small amount of red fibrous tissue, bone trabeculae orderly reconstruction. Compared with the experimental group, the change in the process The control group was slow.
Conclusion: sheep pine lyophilized bone combined with bone marrow mesenchymal stem cells has good osteogenic properties in allogenic metaphysis. However, during the process of osteogenesis, the total amount of bone in 4W decreased significantly, and its biological properties changed.

【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R687;R318.01

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