本文選題：羊　切入點(diǎn)：同種異體　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2014年博士論文　論文類型：學(xué)位論文

【摘要】：第一部羊松質(zhì)凍干骨的制備及生物學(xué)檢測(cè) 目的：制備羊松質(zhì)凍干骨（FDB），研究其大體微觀結(jié)構(gòu)特點(diǎn)，檢測(cè)生物力學(xué)參數(shù)，為羊松質(zhì)凍干骨的應(yīng)用研究提供理論數(shù)據(jù)。 方法：通過(guò)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(jié)構(gòu)；掃描電子顯微鏡（SEM）觀察其超微結(jié)構(gòu)，測(cè)量孔隙大小；微-CT測(cè)量計(jì)算其骨形態(tài)計(jì)量學(xué)參數(shù)； MTS生物力學(xué)機(jī)進(jìn)行力學(xué)性能檢測(cè)，隨機(jī)附帶軟件記錄其最大載荷和應(yīng)力-應(yīng)變曲線，計(jì)算力學(xué)參數(shù)。 結(jié)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(jiàn)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(jié)構(gòu)，孔隙相互連通，測(cè)量孔徑大小為268.60±40.01um。微-CT形態(tài)計(jì)量分析顯示羊松質(zhì)凍干骨的形態(tài)計(jì)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(xué)測(cè)試結(jié)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(qiáng)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(jié)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(xué)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(fù)合間充質(zhì)干細(xì)胞的體外實(shí)驗(yàn)研究 目的：制備骨髓間充質(zhì)干細(xì)胞（BMSCs），研究其生物學(xué)特性、向成骨細(xì)胞分化的能力，及與同種異體松質(zhì)凍干骨復(fù)合的生物相容性。 方法：聯(lián)合密度梯度離心法和貼壁法對(duì)BMSCs進(jìn)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(xì)胞生長(zhǎng)曲線；使用成骨誘導(dǎo)液對(duì)BMSCs進(jìn)行成骨誘導(dǎo)，用茜素紅染色和堿性磷酸酶染色檢測(cè)BMSCs的成骨性能。將第3代BMSCs與羊松質(zhì)凍干骨塊在體外復(fù)合培養(yǎng)。通過(guò)掃描電鏡觀察細(xì)胞在松質(zhì)凍干骨上的附著生長(zhǎng)情況。 結(jié)果：間充質(zhì)干細(xì)胞分離24h后，，部分細(xì)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(xì)胞呈典型的成纖維狀，開(kāi)始形成小的集落，6d后細(xì)胞集落增多，并逐漸連成片狀。10d后各細(xì)胞集落之間逐漸相連，相互融合，鋪滿整個(gè)瓶底。傳代培養(yǎng)的干細(xì)胞懸于培養(yǎng)液中，2h散開(kāi)貼壁，第2d全部貼壁，3-5d時(shí)達(dá)90%。BMSCs的生長(zhǎng)曲線呈S型，P2、P5、P8細(xì)胞的生長(zhǎng)曲線形態(tài)基本一致，第1-2d為潛伏期，生長(zhǎng)較慢；3d后細(xì)胞增長(zhǎng)速度加快，進(jìn)入對(duì)數(shù)期；第7-8d停止生長(zhǎng)，進(jìn)入平臺(tái)期。茜素紅染色檢測(cè)誘導(dǎo)細(xì)胞的分化情況，在12-14d時(shí)出現(xiàn)分化跡象，有點(diǎn)狀的鈣化斑，且分布均勻，16d左右有礦化骨結(jié)節(jié)出現(xiàn)，第21d時(shí)骨結(jié)節(jié)形成比較明顯。堿性磷酸酶染色結(jié)果顯示BMSCs經(jīng)定向誘導(dǎo)21d后出現(xiàn)小的點(diǎn)狀鈣化斑。BMSCs和羊松質(zhì)凍干骨的復(fù)合物（MSCs-FDB）上有細(xì)胞生長(zhǎng)。2h可見(jiàn)細(xì)胞貼附在FDB孔隙內(nèi)部及表面，胞體較小，細(xì)胞與骨小梁結(jié)合不太緊密，隨著培養(yǎng)時(shí)間增加，24h時(shí)胞體增大，生出突觸，與材料結(jié)合緊密。48h時(shí)，細(xì)胞呈不規(guī)則形狀，部分細(xì)胞形成小的集落，細(xì)胞功能活躍，胞體扁平，貼附在小梁骨表面。 結(jié)論：BMSCs具有自我增殖與自我更新能力，P2、P5、P8的細(xì)胞均有很良的生長(zhǎng)性能，P3在成骨誘導(dǎo)劑作用下向成骨細(xì)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(fù)合體成骨行為的體內(nèi)研究 目的：研究MSCs-CFDB復(fù)合體在大動(dòng)物體內(nèi)的成骨行為，為同種異體MSCs-CFDB復(fù)合體在臨床使用提供實(shí)驗(yàn)依據(jù)。 方法：將9只2.5-3年齡40-50kg的實(shí)驗(yàn)綿羊，速眠新Ⅱ注射液麻醉后，在羊脛骨內(nèi)側(cè)距膝關(guān)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(shí)驗(yàn)組)：將培養(yǎng)24h的MSCs-FDB復(fù)合體填充左下肢骨缺損區(qū)。對(duì)照組用DMEM液培養(yǎng)24h的羊松質(zhì)凍干骨植入右下肢相應(yīng)部位骨缺損區(qū)。術(shù)后觀察實(shí)驗(yàn)動(dòng)物的一般活動(dòng)情況及傷口愈合情況；分別術(shù)后4w、8w、12w隨機(jī)各處死3只動(dòng)物，取植骨塊表面軟組織和3cm×3cm×3cm大小骨標(biāo)本，微-CT檢查并重建骨小梁形態(tài)結(jié)構(gòu)，分析測(cè)量主要骨形態(tài)計(jì)量參數(shù)包括骨礦物質(zhì)密度(BMD)、組織骨密度（TMD）、骨體積分?jǐn)?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(jié)果：術(shù)后實(shí)驗(yàn)動(dòng)物活動(dòng)正常，切口正常愈合。大體觀察植骨周圍軟組織，術(shù)后4w見(jiàn)表面纖維組織疏松，淡粉色，于8w、12w時(shí)，逐漸變致密，顏色漸接近周圍正常骨膜組織，無(wú)感染；組織切片未見(jiàn)淋巴浸潤(rùn)。微-CT顯示實(shí)驗(yàn)組與對(duì)照組骨組織結(jié)構(gòu)變化趨勢(shì)一致，術(shù)后4w時(shí)骨小梁結(jié)構(gòu)略增粗，孔隙增大，8w時(shí)小梁結(jié)構(gòu)增粗，12w時(shí)骨小梁結(jié)構(gòu)再次變細(xì)，孔隙增多變小，于所在部位的骨結(jié)構(gòu)整合，不能區(qū)分。微-CT檢測(cè)移植物在4w、8w和12w時(shí)的骨礦物質(zhì)密度BMD：實(shí)驗(yàn)組為145.10±19.96mg/cc，238.41±33.73mg/cc和215.30±28.50mg/cc；對(duì)照組為134.08±23.79mg/cc，240.20±23.02mg/cc和218.70±31.60mg/cc。實(shí)驗(yàn)組的BVF在4w、8w和12w時(shí)分別為0.3772±0.0474，0.6038±0.1099和0.4016±0.0575；對(duì)照組為0.3629±0.0510，0.5967±0.0699和0.4453±0.0503；實(shí)驗(yàn)組和對(duì)照組4w、8w、12w時(shí)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(shí)驗(yàn)組和對(duì)照組的Tb.Sp.在4w、8w、12w時(shí)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(shí)驗(yàn)組和對(duì)照組的Tb.n.在4w、8w、12w時(shí)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(xué)檢查顯示實(shí)驗(yàn)組與對(duì)照組骨組織結(jié)構(gòu)變化趨勢(shì)基本一致，術(shù)后4w時(shí)骨小梁結(jié)構(gòu)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(jiàn)條狀編織骨，原骨小梁組織少部降解,無(wú)炎癥細(xì)胞；8w時(shí)骨小梁結(jié)構(gòu)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(shí)骨小梁結(jié)構(gòu)變細(xì)，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(shì)比單純FDB組更快更明顯。Mason三色染色于術(shù)后4w可見(jiàn)兩組中紅色骨纖維周邊少量藍(lán)綠色骨纖維，8w時(shí)藍(lán)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(xì)胞核明顯。12w時(shí)可見(jiàn)大量藍(lán)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(shí)驗(yàn)組相比，這一變化過(guò)程在對(duì)照組中發(fā)生緩慢。 結(jié)論：羊松質(zhì)凍干骨復(fù)合骨髓間充質(zhì)干細(xì)胞后在同種異體干骺端有較好的成骨性能。但在成骨過(guò)程的4w時(shí)，骨骼總量明顯下降，影響其生物學(xué)性能的組織結(jié)構(gòu)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(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R687;R318.01

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