【作者】 李康杰；

【导师】 孙抒；

【作者基本信息】 延边大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 根据统计,在中国每年大约有一千三百二十万人次发生骨折,其中有10-15%无法愈合,另外,每年因为做关节置换手术、外伤、骨骼发育异常,或是其它原因造成的骨科手术高达一百万例,而在这一百万个手术中,约有四十五万个手术是需要接受骨移植的。然而,骨移植的材料来源不论在数量上或是在质量上,却是明显不足的。骨科医师在临床上,经常碰到的问题,就是骨折的处理,一般人发生骨折之后,只要有适当的处理,大多会如期愈合。在正常愈合所需的时间过后,如果尚未愈合,就是延迟愈合。其原因包括骨折碎片间的间隙过大,固定不可靠,骨折碎片的血液供应不足,开放性骨折合并广泛软组织损伤及感染等等。传统上是解除原因,延长愈合时间持续治疗,但仍然不愈合,就是骨折不愈合。治疗骨折不愈合可用物理性刺激,或是使用骨移植手术的方法。临床上,通常骨移植物来源于自体骨移植：即”取”患者自己身上的骨来”种”到需要的部位,如：髂骨、腓骨或肋骨取下的骨头,移植到需要的部位。一般来说,自体骨移植块的效用最佳,且不会发生疾病传播及排斥反应问题,但由于数量有限,且未能提供够强的大块骨移植块,因此应用范围会受限。而异体骨移植,即取自其它人捐赠骨骼进行骨移植,须经过特殊处理(如低温冰冻、冷冻干燥、辐射处理等),降低其抗原性,以减小排斥现象,并且确定无感染病原后才可使用。但是异体移植骨来源有限,价格昂贵,必须在无菌状态下储存,且保存日期有限,依保存方法而有差异。异体骨移植块的缺点为可能传播肝炎病毒及艾滋病病毒,值得注意。此外,大块异体骨移植块通常会发生骨移植块与宿主骨骼间的融合延缓,以及骨移植块发生吸收或疲乏性骨折等问题。因此,本文研究的重点在于同种异体移植,即将同种骨经过Triton X-100处理后,去除主要的抗原成分,留下矿物质及部分有机成分,作为骨移植块使用。Meezan首先使用化学除垢剂制作了无细胞基底膜,Wilson又首先以Triton X-100为主脱去组织中的所有细胞、抗原、脂质、可溶性蛋白质和可溶性糖胺多糖等物质。其中他采用的除垢剂酶消化方法对狗颈总动脉行脱细胞处理,组织学及电镜观察无细胞成分存在,在基质中主要为胶原和弹性蛋白,结构保存良好。美国Lifecell公司用同样方法制作真皮基质获得了商业性的成功。Dahms对处理的膀胱脱细胞基质成分分析表明了其主要成分为Ⅰ型、Ⅲ型胶原和弹性蛋白。因此,就目前所用的脱细胞处理的方法而言,其基本上保留了促进细胞增殖生长的生物活性成分,为其植入体内修复及再生损伤或缺损的组织提供了物质基础。这样我们有理由相信采用同样方法制备的脱细胞骨基质能够提供渐进式替换作用的骨架,加上自体骨髓间充质干细胞的作用,在体外联合培养后,最终能够修复动物的骨缺损。本研究的主要方法和结果：1、脱细胞骨基质的制备实验联合应用过氧化氢、低渗Tris-Hcl和TritonX-100制备了脱细胞骨基质,之后通过HE染色见新鲜的大鼠松质骨组织切片中可见骨陷窝中含有大量细胞结构,胶原纤维排列整齐；处理组骨陷窝、骨小管中尚有嗜酸性物质残留；处理组标本可见细胞成分基本消失,骨陷窝、骨小管空虚,胶原纤维排列整齐。甲苯胺蓝染色见正常骨松质的骨小梁胶原纤维排列成行,骨细胞占据的骨陷窝分布在骨小梁。骨陷窝中骨细胞核清晰可见,大多数骨陷窝内只有一个骨细胞核。脱细胞骨的细胞外基质胶原纤维排列整齐,染色呈不同程度蓝色,椭圆形骨陷窝内空虚,无骨细胞核及其他结构。2、免疫组化染色免疫组化染色见实验组支架材料的纤维连接蛋白和层粘连蛋白的染色阳性部位在BAECM的骨陷窝周边部,染色较深,呈筛网状、线状分布,为清晰棕色。3、扫描电镜观察扫描电镜可见骨支架是由大量片状骨小梁连接而成的多孔网架,形似海绵状。骨小梁之间的孔隙直径在200-400μm之间,孔间隔厚度为60-100μm。孔隙率为60%,孔洞之间有连联。处理后的骨支架的孔隙内已无骨髓成分。扫描电镜下可见骨小梁上的许多平行排列的骨陷窝,且骨陷窝内已无细胞成分。4、透射电镜观察透射电镜可见脱细胞骨基质的骨陷窝多为低密度影像,有多处空白区,可见少量洗脱残留物质；对照新鲜骨基质的骨陷窝中明显含有细胞成分,并可见细胞核的核仁。免疫组织化学透射电镜可见在脱细胞骨基质的骨陷窝周边弥散存在阳性标记电子密度较高的DAB反应产物呈团块,网状分布,基质可见网状纤维结构,胶原纤维排列整齐。5、洗脱剂残留量测定洗脱剂TritonX-100残留量测定见对照品出峰时间分别为2.495、4.008、4.009min,峰面积948825、1652336、1645545。样品在3min左右无峰值出现。最低检出极限为0.5μl/ml。6、移植免疫分析移植免疫分析在术后4、8周各植入处理组的血清中均可检测出抗体,各时间处理检测组与其相应的无关抗原对照组之间差异非常显著(P<0.01),说明抗体是针对SD大鼠骨抗原的特异性抗体。统计学分析表明：新鲜骨移植组血清抗体相对OD值最高,与脱细胞骨基质差异非常显著(P<0.01)。淋巴细胞刺激实验中经两种移植物第一次体内致敏后,在体外用SD大鼠脾淋巴细胞二次刺激,新鲜骨移植组的淋巴细胞刺激指数最高,与脱细胞骨基质移植组有显著差异(P<0.01),植入SD大鼠两种移植物术后8周,再在体外给予相应移植物二次刺激组中,新鲜骨刺激淋巴细胞增殖,而脱细胞骨基质表现为对淋巴细胞增殖的抑制(P<0.01)。7、骨髓间充质干细胞的培养镜下观察见大鼠BMSCs的原代细胞刚接种时呈圆形,多数悬浮于培养液中,可见克隆形成。48h后大部分细胞都贴壁,换液除去未贴壁的细胞后,可见部分贴壁细胞形态不一,呈梭形的成纤维细胞样或多角形改变,核较大,位于细胞中央或边缘。3-4天开始出现散在的贴壁生长的成纤维样的细胞,再过2-3天,细胞呈现克隆样生长的集落,集落细胞增殖迅速,7天左右每个集落约含有100-200个细胞,细胞形态基本为纺锤形的成纤维细胞样,部分呈宽大扁平的多边形,约9-11天时融合成单层,一般能够达到70%-80%的细胞融合状态。8、流式细胞仪鉴定流式细胞仪检测见培养的第3代BMSCs均一表达CD90、CD44、CD106,阳性率分别为90.05%、95.48%、67.22%;而CD34、CD45、CD11b阴性,阳性率分别为1.02%、0.86%、0.32%。9、诱导后的细胞形态观察倒置显微镜下观察可见多数细胞形态发生明显变化,由梭形转化为圆形或方形,且胞体增大,胞核变大变圆,胞浆中可见较多细小黑色颗粒,胞核色较淡,胞浆色较深,胞核与胞浆对比明显。10、诱导后的细胞鉴定茜素红染色见部分细胞成聚集生长,形成矿化结节,茜素红染色显示为橘红色；钙钻法碱性磷酸酶染色显示胞浆中一些细小黑色颗粒为棕黑色；Ⅰ型胶原免疫荧光染色阳性,红色荧光(Cy3)表示Ⅰ型胶原在细胞内的分布,Ⅰ型胶原多在胞浆靠近胞核处,蓝色荧光(DAPI)为细胞核。11、扫描电镜观察联合培养的脱细胞骨基质与诱导后的成骨样细胞联合培养24,48,72h后,扫描电镜观察并未见脱细胞骨基质组和羟基磷灰石组之间的细胞形态有明显差异,细胞在两组材料上均生长良好,且分布均匀,细胞形态大小不一,但在两组材料中均可见大量的球形细胞黏附在材料上面,每个细胞直径在3-10μm之间,在个别部位还可见正在增殖中的细胞克隆,细胞生长状态良好。12、碱性磷酸酶检测随着培养时间的延长,脱细胞骨基质组和羟基磷灰石组的碱性磷酸酶表达活性都呈上升趋势。在浸提液培养12h和24h,两组之间差异无显著性意义(P>0.05)；在培养48h和72h,脱细胞骨基质组和羟基磷灰石组之间差异有显著性意义(P<0.05)。13、复合细胞的天然脱细胞骨支架修复骨缺损实验成功制作大鼠股骨骨缺损模型后,将复合有细胞的脱细胞骨基质支架材料移植在缺损处,术后4周荧光显微镜见对照组羟基磷灰石支架在镜下显现为黑色条影,周边可见发出绿色荧光的新生骨组织,实验组天然脱细胞骨松质已经能够非常完好地与周边新生骨组织融合在一起,荧光显微镜下无法分辨出其间的差别。普通光镜观察见术后4周及8周的对照组羟基磷灰石显示为黑色条带阴影,而脱细胞骨基质支架之间已填满新生的骨组织,骨支架与新生骨组织很好的融合在一起。偶可见炎性细胞浸润；术后8周甲苯胺蓝染色可见对照组羟基磷灰石显示为黑色条带阴影,而对照组和实验组的支架之间都可以见到已形成的骨单位,新生骨组织已经比较成熟。研究结论：1、利用大鼠股骨成功制备了适于同种异体移植的组织工程骨基质；2、利用动物自身的骨髓间充质干细胞成功地诱导为成骨样细胞；3、制备的脱细胞骨基质在体外适于诱导而来的成骨样细胞的生长；4、联合培养的有细胞的脱细胞骨基质能够很好地修复大鼠股骨骨缺损,有望应用于临床骨缺损疾病的治疗。更多还原

【Abstract】 According to statistics, there are about one thousand and three hundred million people suffer from bone fracture in China every year,10-15%of which won’t be able to be cured.Moreover, the number of orthopedical operation from replacement arthroplasty, trauma,abnormal development of skeleton will be one million or so,about half of which need bone transplantation, but the materials of bone transplantation are insufficient whatever in number or quality. The question that orthopedists often face in most time is to deal with bone fracture in clinic. Most patients suffering from bone fracture will be cured if they are treated in time, but in defined time they aren’t healed which are called delayed union. The reason of poor heal include oversize of interspace of fracture fragments, hypopexia, inadequate blood supply of fracture fragments, open fractures combined with widespread damage of soft tissue and so on. Methods to deal with these things are to keep continuous immobilization, but sometimes the state of patients suffering immobilization being not cured are called disunion. Ways of curing disunion maybe physical stimulation or bone transplantation. So far, most bone transplants originate from autograft, namely get a bone from himself or herself to transplant some required site in himself or herself. Generally speaking, autograft has a best effect on bone transplantation and has no disease transmission in clinic. But the number of autograft is inadequate to supply strong and gross bone mass. So the apply of autograft is limited. While allograft that getting bones from other donors need special handing(e.g. cryogenic freezing, freeze dehydration, irradiation treatment) to reduce the antigen to lower reject reaction.The most disadvantage of allograft is that it possible spread hepatitis virus and human immunodeficiency virus, which deserve paying attention.Therefore, the emphasis of this paper focus on xenograft.Namely, bones of other species are handled by Triton X-100 to get rid of antigen of these bones and leave minerals and some organic principle to apply as bone transplant mass, which are able to supply degradated bone scaffold, which are cultured in vitro with autoallergic bone marrow stromal stem cells(BMSCs) by ways of tissue engineering to be transplanted into the defect of animal bones finally to assess the repair effect. Meezan first used chemical detergents, to isolate morphological and chemical pure basement membranes from several tissues, Then, Wilson use Triton X-100 to get rid of cells, antigen, fat, resoluble protein and glucosamine to produce acellular matrix and apply SEM and microscopic observation to proved that no cells existed in the prepared acellular matrix of small caliber vascular prostheses of dog.Most collegen and plastic protein existed in this kind of matrix.American Lifecell Company used the same method to made a success in business. Dahms’results about acellular gallbladder indicated that type I and III collegen and plastic proteins would existed in the acellular matrx. Therefore, current method kept most bioactive components to promote the growth of cells and provided an effect resolution to repair tissue defect.We believed that used Triton X-100 and other associated drug to make bone scaffolds for tissue engineering to repair animal bone defect with own’s bone marrow mesenchymal stem cells in vitro.Main methods and results:1. Preparation of acellular bone matrixComparing with flesh spongy bone, appearance of ANECM is more shallow and white. We also use different concentration of TritonX-100 and time of cell extraction and Prove 30 days of cell extraction and 3%TritonX-100 to be most effective. Cell component isn’t found under light and electron microscope. Under light microscope, collagen fibers of arrange uniformly. It’s blankness in ellipse bone lacunas and there is no nucleus and other configuration. Osteocytes are in focus under scanning electron miscroscope of flesh spongy bone. No osteocyte and remnent is observed in ANECM. The wall of bone lacuna is smooth and regular. We surveyed the density and other parameters of histomorphology of bone lacunas. Those parameters have important significance to measure the bulk of osteocytes. We may estimate the cell number per volumn of bone tissue and provide the foundation for construction of engineering bone.2. Immunohistochemical stainingLN and FN dyeing submit bolt and line shape in bone matrix around bone lacuna. Distinct brown provides negative. Negative comparisons have not brown granules.There are positive markers around bone lacuna and because high electron density distribution of DAB reaction production submits block and reticulation.3. SEM observationScanning electron microscope displayed that different layers of acellular natural bone matrix connected loosely and presented amounts of pore space between layers.4. TEM observationTransmission electron microscope showed that no high density image appeared in lacuna area.5. Residul test of chemical detergentsApexes of comparition sample in result are 2.495,4.008,4.009min, and apex areas are 946825,1652336,1645545. Sample of bone acellular extra-cell matrix has not apex about 3min. The lowest examination limit is 0.5μl/ml.6. Transplantation immunological analysisLymphocyte stimulation assay showed that immunological rejection of flesh bone was much greater than that of acellular natural bone matrix (p< 0.01).7. Culture of bone marrow mesenchymal stem cellsThe primary cells and the passage cells were mostly fusiform in shape, to be similar to fibroblast cell.8. Analysis of flow cytometrThe flow cytometer analysis showed that the membrane mark CD44, CD90,CD106 were positive, CD34,CD45,CDllb were negative.9. Observation of the induced cellsMost induced cells changed into round cells and square cells in shape,and their body became larger and many minite black pellet present in the cytoplasm of these cells.10. Identity of induced cellsAfter BMSCs were induced, cell alizarin carmine staining showed that it was positive in osteogenetic cell, and ALP staining showed that black granules existed in induced cells. Immunohistochemisty staining showed that type I collagen existed in cytoplasm.11. SEM observation of acellular extra-cell matrix with induced cellsScanning electron microscopy displayed that osteoblast-like cells on the surface of acellular natural bone matrix aggregated together and grew as three-dimensional style.12. ALP detectionAlkaline phosphatase detection kit analysis indicated that acellular natural bone matrix prepared with TritonX-100 were able to promote the growth of osteoblast-like cells compared with calcium hydroxyapatite after 48 hours of culture.13. Experiment on bone acelluar extra-cell matrix with induced BMSCs repair-ing aminal bone defectBoth bone acelluar extra-cell matrix and calcium hydroxyapatite combined with osteoblast-like cells from BMSCs were implanted into the femural defect of rats after the defect models were made successfully. At the 4th week, fluorescence microscope showed that bar-like calcium hydroxyapatite and new bone with green fluorescence appear in scope of eyes in control group. Compared with the control group, experimental bone acelluar extra-cell matrix fused with peripheral new bone and we couldn’t tell the differences with each other under flurescence microscope. At the 8th week, toluidine blue staining showed that osteon in new bone have formed whatever in control or test group.Conclusions:1. Prepare bone acelluar extra-cell matrix successfully.2. BMSCs were able to differentiate into osteoblast-like cells after the conditonal fluids was added.3. Prepared bone acelluar extra-cell matrix fit for the induced cells in vitro.4. Bone acelluar extra-cell matrix with osteoblast-like cells are able to repair femural defect of rats successfully.更多还原