【作者】 万军；

【导师】 张湘生；

【作者基本信息】 中南大学 ， 外科学， 2011， 博士

【摘要】 第一章自体血清培养人骨髓间充质干细胞的可行性评估目的：胎牛血清体外培养的人骨髓间充质干细胞增加体内移植的风险,限制其临床应用。本部分实验旨在探讨自体血清体外培养人骨髓间充质干细胞的可行性。方法：全骨髓贴壁培养法分离、纯化人骨髓间充质干原代细胞,取第三代细胞按加入培养血清的不同分为胎牛血清培养组和自体血清培养组。倒置相差显微镜观察两组培养细胞的生长情况；使用MTT法测定两组培养传代第3代细胞和第5代细胞的增殖情况,绘制细胞增殖曲线；取两组培养传代第3代细胞行成软骨细胞、成骨细胞及成脂肪细胞诱导分化,并分别对成软骨细胞行甲苯胺蓝染色、Ⅱ型胶原染色；对成骨细胞行碱性磷酸酶(ALP)染色、VonKossa染色；对成脂肪细胞行油红O染色,并计算两组细胞诱导分化阳性率；取自体血清培养组传代第5代细胞行流式细胞仪测定鉴定细胞表面CD44. CD90抗原表达情况。结果：倒置相差显微镜下观察全骨髓贴壁法分离纯化的原代细胞形态均一,呈长梭形,两组培养细胞近融合状态均呈漩涡样同向生长特征；MTT测定两组培养第3代及第5代细胞均具备良好的生长增殖潜能,第3-6d是增殖对数增长期,两组细胞增殖无统计学差异；两组培养第3代细胞均能在特定诱导环境下向成软骨细胞、成骨细胞及成脂肪细胞分化,成软骨细胞经甲苯胺蓝染成蓝紫色,Ⅱ型胶原染成黄色；成骨细胞碱性磷酸酶染色(ALP)阳性,VonKossa钙结节染成黑色；成脂肪细胞内经油红O染成红色脂滴,两组诱导分化细胞阳性率无统计学差异；自体血清培养组第5代细胞经流式细胞仪鉴定细胞表面不表达CD34、CD45,而表达CD44、CD90。结论：全骨髓贴壁培养法操作简单,细胞纯化率高；自体血清培养组细胞保持了骨髓间充质干细胞典型的生长特征、增殖以及诱导分化潜能；自体血清培养组细胞表面不表达造血干细胞表面抗原,而表达骨髓间充质干细胞表面抗原,符合骨髓间充质干细胞特征。第二章CPC/FG/OM复合材料的研制及理学性质测定目的：股骨头坏死是临床治疗上颇为棘手的问题之一。有研究表明坏死股骨头软骨下骨板的支撑是治疗股骨头坏死的关键因素。磷酸钙骨水泥(calcium phosphate cement, CPC)材料是一种兼备修复与成型的骨组织修复材料,具备自固化与骨传导特性。但是由于材料本身的缺陷存在抗压强度低,无骨诱导活性等缺点,限制了其在人体负重区域的临床应用。纤维蛋白胶(fibrin glue, FG)是临床上广泛应用的粘合剂,与CPC复合能够利用其粘性增加CPC的抗压强度。成骨诱导剂(osteogenesis medium, OM)已被证实对骨髓间充质干细胞有着诱导成骨分化的作用。本部分实验旨在研制一种新型的适用于治疗股骨头坏死的填充材料—CPC/FG/OM复合材料,以期复合材料即能够保留单纯CPC材料自固化及优良骨传导性的优点,又能够通过加入FG增强材料的抗压强度以满足人体负重区域抗压强度要求以及加入OM增加材料的成骨诱导活性以改善坏死股骨头内的成骨内环境。方法：将OM加入FG中制备FG/OM溶液,按照3：1的粉末/液体比例配置CPC/FG/OM复合材料,按照相同的粉末/液体比值配置CPC/FG复合材料及CPC材料作为两组对照组。对三组材料进行扫描电镜观察材料表面结构；XRD晶体分析；凝固时间(吉尔摩针法)、抗压强度、弹性模量(MTS S10力学性能实验机测试)以及孔隙率的测定(阿基米德法)。结果：三组材料扫描电镜观察发现单纯CPC材料表面孔隙分布均匀,孔隙较小,而CPC/FG及CPC/FG/OM两种复合材料表面孔隙分布不均匀,孔隙尺寸大；XRD分析显示三种材料的XRD曲线与羟基磷灰石相似；CPC/FG及CPC/FG/OM两种材料初凝时间[(6.8±0.5)min,(7.28±0.6)min]明显长于CPC材料[(3.38±0.4)min],差异有统计学意义(P<0.05),三种材料终凝时间差异无统计学差异(P>0.05); CPC/FG及CPC/FG/OM两种材料抗压强度及弹性模量[(8.94±0.5)MPa,(9.1±0.4) MPa;(2.73±0.07)GPa,(2.846±0.1)GPa]明显高于CPC材料组[(5.08±0.5)MPa,(1.064±0.14)GPa],差异有统计学意义(P<0.05)；CPC/FG及CPC/FG/OM两种材料孔隙率[(38.64±1.5)%,(37.9±1.4)%]明显低于CPC材料组[(48.82±8.2)%],差异有统计学意义(P<0.05)。结论：三种材料晶体相与羟基磷灰石一致,证实体内降解产物与人体骨骼无机成分相同；CPC/FG/OM复合材料中OM的加入没有影响其理学性质：凝固时间满足临床经皮穿刺的可注射性要求；抗压强度及弹性模量与CPC/FG复合材料相仿,明显高于CPC材料,达到坏死股骨头软骨下骨板的填充材料抗压强度要求；孔隙率与CPC/FG复合材料相仿,明显低于CPC材料,但是由于其表面呈大尺寸孔隙特征,有利于种子细胞早期帖附。第三章CPC/FG/OM复合材料与hBMSCs体外共培养的实验研究目的：股骨头坏死的治疗涉及到重建生物力学坚强(坏死股骨头软骨下骨板的支撑)和重建生物学坚强(坏死股骨头内源性的修复)。目前骨组织工程以及干细胞工程的兴起使得骨髓间充质干细胞移植成为未来治疗股骨头坏死的新方向。实验二已经证实CPC/FG/OM复合材料满足即时重建坏死股骨头生物力学坚强要求,本部分实验将CPC/FG/OM复合材料与自体血清培养的hBMSCs共培养,探讨CPC/FG/OM复合材料的生物相容性和成骨诱导性能。方法：三组材料按实验二步骤制备。hBMSCs按实验一步骤准备。将三种材料各放置入24孔板,hBMSCs接种。扫描电镜观察细胞在三组材料上的生长粘附情况；MTT法检测培养第1至7d细胞在三组材料上的生长增殖情况；RT-PCR检测细胞在三组材料中培养7d后的碱性磷酸酶(ALP)、Ⅰ型胶原蛋白(COL-I)、骨钙素(OCN)mRNA的表达水平；并分别检测细胞在三组材料中培养第3d,第7d,第14d碱性磷酸酶活性(pNPP法)以及骨钙素含量(ELISA法)。结果：扫描电镜观察细胞在三组材料上均可以粘附生长,CPC组细胞间连接较少,CPC/FG和CPC/FG/OM两组细胞间连接多,细胞外基质分泌较多；MTT法检测细胞在CPC/FG和CPC/FG/OM两组增殖较CPC组增殖快；碱性磷酸酶(ALP)、Ⅰ型胶原蛋白(COL-I)、骨钙素(OCN)在CPC/FG/OM组表达最强,CPC/FG组次之,CPC组最弱。结论：CPC/FG, CPC/FG/OM以及CPC/FG/OM复合材料生物相容性均良好,但是CPC/FG/OM复合材料诱导成骨能力明显高于CPC/FG组与CPC组。第四章经皮穿刺CPC/FG/OM复合材料股骨头填充联合hBMSCs移植治疗股骨头坏死的动物实验研究目的：通过动物实验观察经皮穿刺CPC/FG/OM复合材料股骨头填充联合hBMSCs移植治疗股骨头坏死的效果方法：采用激素加内毒素法建造新西兰大白兔股骨头坏死动物模型(耳缘静脉注射大肠杆菌内毒素(10ug/kg),并同时在兔臀部肌肉注射甲基泼尼松龙(40mg/kg),共注射3次,每次间隔24h),取股骨头坏死造模成功的24只大白兔随机分为四组,A组：经皮穿刺组(单纯减压组)；B组：经皮穿刺+hBMSCs移植组；C组：经皮穿刺CPC/FG/OM复合材料股骨头填充组；D组：经皮穿刺CPC/FG/OM复合材料股骨头填充+hBMSCs移植组。其中每组6只,一侧行手术操作,对侧不进行任何操作作为对照组。在术后第4、8、12w进行影像学评估(x-ray检查评估股骨头坏死转归情况)和组织学评估(HE染色、软骨下骨区域成骨细胞计数和陷窝面积百分比计算)。结果：A组,B组与对照组比较治疗效果不明显,影像学评估与对照侧无明显差别,早期出现股骨头坏死进展迹象(软骨下骨囊性变区域增多,关节间隙变窄),组织学大体观察示股骨头多处凹陷区域,股骨头色泽变白,HE染色显示坏死股骨头软骨下骨区域少见成骨细胞及骨基质形成,12w软骨下骨区域成骨细胞计数和陷窝面积百分比计算与对照侧比较差异无统计学意义；C组和D组与A组、B组及对照组比较治疗效果明显,12w影像学评估两组治疗侧股骨头圆形外观保持良好,未见股骨头囊性变区域,C组材料影清晰,与骨界限清楚,D组材料影模糊,与骨界限不清楚,周围有新骨生成影像。组织学大体观察示C组治疗侧股骨头色泽发白,D组色泽正常,HE染色示C组较D组髓内新骨基质形成较少,成骨细胞少。治疗12w软骨下骨区域成骨细胞计数和陷窝面积百分比计算与D组比较差异有统计学意义。结论：动物实验结果显示经皮穿刺CPC/FG/OM复合材料填充联合hBMSCs移植治疗股骨头坏死治疗效果明显,优于髓芯减压组,hBMSCs移植组,单纯CPC/FG/OM复合材料填充组。更多还原

【Abstract】 Chapter I Evaluation of feasibility of human bone marrow mesenchymal stem cell cultured by autologous serumObjective:Translations of human bone marrow mesenchymal stem cell cultured by fetal bovine serum have various risks which limit its clinical application. The experiment was to investigate the feasibility of establishing a suitable hBMSCs lines for clinical application cultured by autologous serum instead of fetal bovine serum in vitro.Methods:The hBMSCs was isolated using whole bone marrow adherent way. The passage3of hBMSCs was devided as two groups according the different culturing serum:group of fetal bovine serum and group of autologous serum. The growth of cells were observed by using inverted phase contrast microscope; the MTT method was used to measured the proliferation of passage3and5, then the results were turned to the cell proliferation curve drawing; the passage3of two groups were taken to differentiating into cartilage cells (toluidine blue staining and collagen Ⅱ staining), osteoblasts cells (alkaline phosphate staining and VonKossa staining) and fat cells (oil red O staining) respectively, the positive rates of cell differentiation of two group were calculated; the passage5of autologous serum group was identified CD34,C44,CD45and CD90antigen expression by flow cytometry.Results:The hBMSCs purified by whole bone marrow adherent way was observed taking a elongated spindle presentation under inverted phase contrast microscope. Cells of two groups showed a spiral-like matter of growth in near fusion. The results of MTT show that the passage3and5of two groups possessed a good potential for growth and proliferation. The period of logarithmic growth phase was3-6d. There is no statistic difference between the two groups. hBMSCs of two groups can be differentiated into cartilage cells(stained purple by toluidine blue and yellow by collagen II), osteoblasts(stained ALP positive and lack in cell nuclear by VonKossa) and fat cells(stained red lipid droplets by oil red O), the positive rate of cell differentiation show no significant statistic difference between two groups; the passage5cultured by autologous serum was identified the CD44and CD90antigen expression by flow cytometry without CD34and C45expression. Conclusions:Whole bone marrow adherent culture way is simple and has high purification rate of hBMSCs. The hBMSCs cultured by autologous serum maintained the typical growth characteristics and potentials of proliferation and differentiation of stem cells. The hBMSCs of autologous serum group did not express hematopoietic stem cell surface antigen but BMSCs surface antigen which fit the BMSCs characteristics. Chapter II Preparation of CPC/FG/OM composite material and evaluation of its physical propertiesObjective:The clinical treatment of femoral head osteonecrosis remains a tough issue. Researches have shown that supporting the subchondral bone of femoral head is a key factor for treatment. Calcium phosphate cement (CPC) is a bone tissue material combined repairing and building, which have properties of self-setting and bone conduction. However, due to defects of CPC materials, CPC has some disadvantages such as low strength and lack of bone inducing activity which limit its clinical application especially in weight-bearing area of human body. Fibrin glue(FG) is widely used in clinical as a excellent adhesives, which can increase compressive strength of the CPC by using its viscosity. Osteogenesis medium(OM) has been proved having capability to differentiate stem cells into osteoblasts. The experiment is to evaluate physical property of a novel material, CPC/FG/OM composite material, for treatment of femoral head osteonecrosis. The novel material was expected to remain properties of self-setting and bone conduction of CPC, along with increased compressive strength by adding FG to fulfill the requirement for support the subchondral bone of femoral head and property of bone induction by adding OM to fulfill the requirement for improving the osteogenic activity in femoral head osteonecrosis.Methods:OM was added into FG to making FG/OM solution. According to a3:1power/liquid ratio, CPC powder was mixed with FG/OM solution to making CPC/FG/OM composite materials. CPC/FG material and CPC material was made as the same powder/liquid ratio and regarded as two control groups. Evaluation the physical properties include scanning electron microscopy observation; XRD analysis; setting time (Gilmore needle method); compressive strength and elastic modulus(MTS S10mechanical properties testing machine) and porosity(Archimedes method).Results:Uneven pore distribution and large size pore were observed in both CPC/FG and CPC/FG/OM group comparing with smooth distribution and small size pore in CPC group(SEM). The result of XRD analysis find that the three group has similar crystal phase to hydroxyapatite; the initial setting time of CPC/FG and CPC/FG/OM [(6.8±0.5)min,(7.28±0.6)min]were significantly longer than the CPC group[(3.38±0.4) min](there is significant statistic differences, P<0.05); the final setting time of three group was similar, there is no significant statistic difference among them(P>0.05); the compressive strength and elastic modulus of CPC/FG and CPC/FG/OM [(8.94±0.5)MPa,(9.1±0.4) MPa;(2.73±0.07)GPa,(2.846±0.1)GPa] were significantly higher than the CPC group[(5.08±0.5)MPa,(1.064±0.14)GPa](there is significant statistic differences,P＜0.05); the porosity of CPC/FG and CPC/FG/OM [(38.64±1.5)%,(37.9±1.4)%]were significantly lower than the CPC group[(48.82±8.2)%](there is significant statistic differences, P<0.05).Conclusions:The three groups have similar crystal phase to hydroxyapatite which means the degraded products in vivo is the same as inorganic constitutes of human bone. Adding OM did not influence the physical property of CPC/FG material:setting time reach the property of injectability when applying percutaneous clinical operation; compressive strength and elastic modulus reach the requirement of support the subchondral bone of femoral head; despite of lower porosity, the surface of CPC/FG/OM have large size pore which can easier for early attachment of seed cells. ChapterⅢ Evaluation of autologous serum cultured hBMSCs seeding on CPC/FG/OM composite materialObjective:There are two key elements need to be concerned in treatment of femoral head osteonecrosis:rebuilding both the biomechanical strength (supporting the subchondral bone) and biological strength (autologous repairing) of femoral head. The development in both bone tissue engineering and stem cell engineering make it a new direction for treatment of femoral head osteonecrosis-translation of stem cells. Study in chapter II has proved that CPC/FG/OM composite material can be an ideal material to rebuild biomechanical strength of femoral head immediately. This chapter aims to evaluate the biocompatibility and property of bone induction of CPC/FG/OM composite material by culturing autologous serum-cultured hBMSCs with material.Methods:Preparations of materials were according to same methods described in chapter Ⅱ. Autologous serum-cultured hBMSCs was prepared in ways described in chapter I. Three materials were put into three separated24-well plate with hBMSCs seeding. SEM (scanning electron microscope) was used to observe the growth of cells in three materials. The proliferation of cell were measured by MTT method in the period of1to7-day respectively; mRNA expression of ALP, COL-I and OCN were measured by RT-PCR method; and activity of ALP(pNPP kit) and content of OCN(human OCN ELISA kit) were measured in the period of3-day,7-day and14-day respectively.Results:hBMSCs were observed in good shape and lots of cell-cell junction in both CPC/FG and CPC/FG/OM group with less junction in CPC group(SEM observation). The proliferations of hBMSCs were significant increased in both CPC/FG and CPC/FG/OM group. The expression of mRNA of ALP, COL-I and OCN was highest in CPC/FG/OM group with CPC/FG lower and CPC lowest. The acticities of ALP were highest in CPC/FG/OM group with CPC/FG lower and CPC lowest. The content of OCN was positive in CPC/FG/OM group after3-day culturation; however it is positive until post-14-day culturation in CPC and CPC/FG group.Conclusions:CPC/FG/OM composite material has excellent biocompatibility and strong property of bone induction comparing with CPC/FG group and CPC group. Chapter IV Animal studies on percutaneous CPC/FG/OM composite material filling combined with translation of hBMSCs in treatment of femoral head osteonecrosisObjective:Evaluating the effect of percutaneous CPC/FG/OM composite material filling combined with translation of hBMSCs on treatment of femoral head osteonecrosis through animal studies.Methods:The hormonal combined endotoxin injection method was chosen to establish New Zealand white rabbit model of femoral head osteonecrosis.(Ear vein injection of Escherichia coli endotoxin (lOug/kg) combined with intramuscular injection methyl-prednisolone (40mg/kg in New Zealand white rabbits). After the animal model have been successfully established,24rabbits were randomly divided into four groups:group A: percutaneous group(simple decompression group); group B:percutaneous hBMSCs translation group; group C:pertaneous CPC/FG/OM filling group and group D:percutaneous CPC/FG/OM filling combined with translation of hBMSCs group. Every group has six rabbits. One side was operated and the contralateral side was regarded as the control group with no operation taken. Radiology and histology evaluation were made every4w,8w and12w.Results:There are no statistic difference among group A,B and control group, radiology exam show that cystic area in subchondral area increased in early stage with narrowing joint space of operation side. The gross view of femoral head show many pit in the femoral head which turn to be a white color in both group, HE staining show that rare intramedullary osteoblasts in the subchondral bone of femoral head and few bone matrix formation, there are no statistic difference about cell count of intramedullary osteoblasts and percentage of bone resorption area among this three group in12w; group C and D have a good effect treatment. Radiology exam show the round shape of femoral head remained in12w and found no cystic lesion in subchondral area of femoral head. Group C has a clearer image of material than group D which has new bone formation image around the material. Gross view of femoral head show white color in group C comparing with the normal color in group D. HE staining show more osteoblasts and new bone matrix formation in group D. More cell count of osteoblasts and less percentage of bone resorption in subchondral area was observed in group D comparing with group C in12w.Conclusions:Results of the animal study show that percutaneous CPC/FG/OM composite materail filling combined with translation of hBMSCs has a significant effect on dealing with femoral head osteonecrosis, compared with core decompression, hBMSCs translation and CPC/FG/OM filling method.更多还原