羊同种异体松质冻干骨生物学行为的实验研究

发布时间：2018-03-22 04:03

本文选题：羊　切入点：同种异体　出处：《中国人民解放军医学院》2014年博士论文　论文类型：学位论文

【摘要】：第一部羊松质冻干骨的制备及生物学检测目的：制备羊松质冻干骨（FDB），研究其大体微观结构特点，检测生物力学参数，为羊松质冻干骨的应用研究提供理论数据。方法：通过脱脂脱蛋白及冷冻辐照等方法制备羊松质冻干骨和未冻干的深冻骨。观察大体形态和结构；扫描电子显微镜（SEM）观察其超微结构，测量孔隙大小；微-CT测量计算其骨形态计量学参数； MTS生物力学机进行力学性能检测，随机附带软件记录其最大载荷和应力-应变曲线，计算力学参数。结果：制备成大小为10×10×10mm3羊髂骨松质冻干骨块及深冻骨块。大体观察见羊松质冻干骨块外观呈乳白色，透亮，扫描电子显微镜下羊松质冻干骨块呈三维多孔网状结构，孔隙相互连通，测量孔径大小为268.60±40.01um。微-CT形态计量分析显示羊松质冻干骨的形态计量参数，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。力学测试结果：冻干骨与深冻骨的最大抗压力分别是663.57±20.98Nu和430.41±31.47Nu；最大压缩强度分别为8.19±0.80MPa和5.31±0.62MPa；弹性模量分别为289.45±30.10MPa和23.37±3.45MPa。结论：羊松质冻干骨具有作为支架材料的理想孔隙大小及孔隙率，有一定的生物力学抗压性能，是一种理想的骨组织工程支架材料。第二部分羊松质冻干骨复合间充质干细胞的体外实验研究目的：制备骨髓间充质干细胞（BMSCs），研究其生物学特性、向成骨细胞分化的能力，及与同种异体松质冻干骨复合的生物相容性。方法：联合密度梯度离心法和贴壁法对BMSCs进行分离及传代培养。取第2、5、8代的BMSCs培养观察并绘制细胞生长曲线；使用成骨诱导液对BMSCs进行成骨诱导，用茜素红染色和碱性磷酸酶染色检测BMSCs的成骨性能。将第3代BMSCs与羊松质冻干骨块在体外复合培养。通过扫描电镜观察细胞在松质冻干骨上的附着生长情况。结果：间充质干细胞分离24h后，，部分细胞贴壁，多以圆形、椭圆形等不规则形态散在分布，4d后细胞呈典型的成纤维状，开始形成小的集落，6d后细胞集落增多，并逐渐连成片状。10d后各细胞集落之间逐渐相连，相互融合，铺满整个瓶底。传代培养的干细胞悬于培养液中，2h散开贴壁，第2d全部贴壁，3-5d时达90%。BMSCs的生长曲线呈S型，P2、P5、P8细胞的生长曲线形态基本一致，第1-2d为潜伏期，生长较慢；3d后细胞增长速度加快，进入对数期；第7-8d停止生长，进入平台期。茜素红染色检测诱导细胞的分化情况，在12-14d时出现分化迹象，有点状的钙化斑，且分布均匀，16d左右有矿化骨结节出现，第21d时骨结节形成比较明显。碱性磷酸酶染色结果显示BMSCs经定向诱导21d后出现小的点状钙化斑。BMSCs和羊松质冻干骨的复合物（MSCs-FDB）上有细胞生长。2h可见细胞贴附在FDB孔隙内部及表面，胞体较小，细胞与骨小梁结合不太紧密，随着培养时间增加，24h时胞体增大，生出突触，与材料结合紧密。48h时，细胞呈不规则形状，部分细胞形成小的集落，细胞功能活跃，胞体扁平，贴附在小梁骨表面。结论：BMSCs具有自我增殖与自我更新能力，P2、P5、P8的细胞均有很良的生长性能，P3在成骨诱导剂作用下向成骨细胞定向分化，与同种异体松质冻干骨组织相容性好。第三部分羊MSCs-CFDB复合体成骨行为的体内研究目的：研究MSCs-CFDB复合体在大动物体内的成骨行为，为同种异体MSCs-CFDB复合体在临床使用提供实验依据。方法：将9只2.5-3年龄40-50kg的实验绵羊，速眠新Ⅱ注射液麻醉后，在羊胫骨内侧距膝关节面下3-5mm处，截骨形成10×10×10mm3干骺端骨缺损模型。A组(实验组)：将培养24h的MSCs-FDB复合体填充左下肢骨缺损区。对照组用DMEM液培养24h的羊松质冻干骨植入右下肢相应部位骨缺损区。术后观察实验动物的一般活动情况及伤口愈合情况；分别术后4w、8w、12w随机各处死3只动物，取植骨块表面软组织和3cm×3cm×3cm大小骨标本，微-CT检查并重建骨小梁形态结构，分析测量主要骨形态计量参数包括骨矿物质密度(BMD)、组织骨密度（TMD）、骨体积分数（BVF）、骨小梁厚度(Tb.Th.)、骨小梁间隙宽度(Tb.Sp.)和骨小梁数量(Tb.n.)。脱钙后行HE染色和Masson三色染色，分析植入物的成骨情况。结果：术后实验动物活动正常，切口正常愈合。大体观察植骨周围软组织，术后4w见表面纤维组织疏松，淡粉色，于8w、12w时，逐渐变致密，颜色渐接近周围正常骨膜组织，无感染；组织切片未见淋巴浸润。微-CT显示实验组与对照组骨组织结构变化趋势一致，术后4w时骨小梁结构略增粗，孔隙增大，8w时小梁结构增粗，12w时骨小梁结构再次变细，孔隙增多变小，于所在部位的骨结构整合，不能区分。微-CT检测移植物在4w、8w和12w时的骨矿物质密度BMD：实验组为145.10±19.96mg/cc，238.41±33.73mg/cc和215.30±28.50mg/cc；对照组为134.08±23.79mg/cc，240.20±23.02mg/cc和218.70±31.60mg/cc。实验组的BVF在4w、8w和12w时分别为0.3772±0.0474，0.6038±0.1099和0.4016±0.0575；对照组为0.3629±0.0510，0.5967±0.0699和0.4453±0.0503；实验组和对照组4w、8w、12w时的Tb.Th.分别为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；实验组和对照组的Tb.Sp.在4w、8w、12w时分别为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。实验组和对照组的Tb.n.在4w、8w、12w时分别为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。组织学检查显示实验组与对照组骨组织结构变化趋势基本一致，术后4w时骨小梁结构稀疏，略增粗，孔隙增大，有新纤维骨生成，在原骨小梁的周边可见条状编织骨，原骨小梁组织少部降解,无炎症细胞；8w时骨小梁结构增粗，小梁变密，新骨的形成及移植骨的降解都明显增加，出现新骨旧骨的相互交织状态；12w时骨小梁结构变细，孔隙增多变小，原骨小梁组织大部分被取代，新骨逐渐向成熟骨组织转变。总体上，MSCs-FDB组的变化趋势比单纯FDB组更快更明显。Mason三色染色于术后4w可见两组中红色骨纤维周边少量蓝绿色骨纤维，8w时蓝绿色新生骨纤维组织增多，与红色纤维组织骨相互交织包绕，组织内细胞核明显。12w时可见大量蓝绿色新生骨组织，有少量红色纤维组织，重建的骨小梁规整。与实验组相比，这一变化过程在对照组中发生缓慢。结论：羊松质冻干骨复合骨髓间充质干细胞后在同种异体干骺端有较好的成骨性能。但在成骨过程的4w时，骨骼总量明显下降，影响其生物学性能的组织结构发生变化。
[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.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R687;R318.01

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