bBMP异位诱导成骨的Micro—CT评价

借助Micro—CT评价单纯牛骨形态发生蛋白（bovine bone morphogenetic protein,bBMP）异位诱导成骨的长期三维影像学及骨质变化。（20±2）g昆明小鼠21只,麻醉后于双侧股部肌肉中植入bBMP各2mg,分别于1、2、4、6、8、10、12周各处死3只,切取诱导分化组织,5％戊二醛固定,行Micro—CT扫描和三维重建,运用ABA专用骨骼分析软件测定组织矿含量（tissue mineral content,TMC）,组织骨密度（tissue mineral density,TUB）,骨体积分数（bone volume fraction,BVF）,结构模型指数（structure model index,SMI）,骨小梁厚度（trabecular thickness,Tb．Th）,骨小梁数量（trabecular number,Tb．N）及皮质骨骨密度（bone mineral density,BMD）等参数,运用SPSS10．0统计软件进行统计学分析。bBMP从植入2周开始逐渐形成一椭圆形骨组织块,2～4周,异位生成骨呈疏松的新生骨,4周时组织矿含量达第一个峰值,骨小梁数量最多;随着观察时间的延长（6-12周）,异位诱导生成的椭圆形骨组织内部骨小梁逐渐吸收,数量减少,12周时骨小梁数量最少;而外层骨组织逐渐塑形成为皮质骨,12周时骨矿含量值、骨小梁厚度、组织骨密度和皮质骨骨密度均达最大值。说明bBMP具有强大的异位骨诱导能力,血供不足时,骨质降解吸收;血供充足时,骨质逐渐成熟改建。     

bBMP异位诱导成骨的组织学长期观察

目的长期观察并获得单纯牛骨形态发生蛋白(bovine bone morphogenetic protein,bBMP)异位诱导成骨的组织学变化。方法(20±2)g昆明小鼠21只,麻醉后于双侧股部肌肉中直接植入块状bBMP各2mg,分别于第1、2、4、6、8、10、12周各处死3只,切取诱导分化组织,5%戊二醛固定,标本用10%EDTA脱钙后制作5μm石蜡切片,分别行甲苯胺蓝染色,HE染色,丽春红三色染色观察组织学变化情况。结果植入1周,大量分化良好的间充质细胞和幼稚软骨细胞形成一椭圆形分化组织块;植入2周,大量骨小梁生成,部分骨小梁边缘骨化;植入4周,骨小梁转化为成熟的骨质,骨质中有散在于骨细胞中的破骨细胞生成;植入6~12周,椭圆形骨组织块内部小梁骨逐渐吸收、断裂、不连接,但外周骨质逐渐塑形,局部成熟骨质周边仍伴有新生骨形成。结论bBMP无需任何载体即可在肌肉中异位成骨,具有强大的异位骨诱导能力;血供在异位骨的形成和维持中可能起着重要作用。     

Micro-CT分析跟骨内骨小梁分布及结构特征

背景：跟骨位于足的后下部,受力较大。跟骨外形复杂且不规则,外周有较薄的皮质骨包绕,内部充满大量的骨小梁,探讨骨小梁的微观结构、走行及分布特征有助于提高对跟骨骨折的认识。目的：采用微计算机断层扫描仪(Micro-CT)扫描跟骨标本获得其影像资料,对其内部骨小梁结构进行分析,探讨跟骨内骨小梁的形态、分布及结构特征。方法：采用Micro-CT连续扫描干燥成人跟骨标本,扫描后获得影像图像,将其以DICOM格式存储。导入影像资料到Hiscan Analyzer软件,显示清晰完整的成人跟骨矢状面、冠状面、水平面的图像,逐层观察骨小梁的走行,依据骨小梁走行特点将跟骨矢状面分为6个部分,在以7 mm为标准的相同厚度下,每个部分选择1个49 mm²等面积的兴趣区,三维重建后获得跟骨及骨小梁的立体微观结构,二值化后运用软件计算感兴趣区域内骨小梁的体积分数、表面密度、骨小梁厚度、骨小梁间隙和骨小梁数量参数。结果与结论：(1)跟骨表面皮质层很薄,内部充满大量骨松质,在Gissane角的骨皮质明显增厚;(2)跟骨上部的骨小梁体积分数大于跟骨下部前端、中和三角区、跟骨下后部、跟骨结节部、跟骨...     

骨形态发生蛋白及其活性影响因素探讨

骨形态发生蛋白（ＢＭＰｓ）是骨和软骨诱导形成的分子调节器,它调节着骨母细胞的 移、增殖、公化,对它的研究会加速骨组织工程的进展。研究表明,ＢＭＰｓ是一组复合体,它家族中的第一个成员以及复合体均有诱民成骨的活性,但其分子形态、浓度、所处的微环境等都影响着它的骨效应。本文简述了它的结构,并重点搪塞了在骨组织工程研究中影响ＢＭＰｓ活性的因素。     

腺病毒载体介导的BMP-2转基因诱导成骨研究进展

骨形态发生蛋白2可诱导具有成骨分化潜能的间充质细胞向成骨细胞分化。腺病毒载体介导的BMP-2转基因方法被认为是最有效的BMP-2转基因诱导成骨手段。本文参考了大量的有关英文文献，从BMP-2腺病毒载体及其介导的BMP-2转基因基本途径、成骨诱导研究所取得的成果和存在的问题等几个方面，重点回顾了近五年来的研究进展，并对这一领域的前景进行了一些展望。     

LPS激活TLR4抑制BMP9诱导iMEFs成骨分化

目的研究脂多糖(LPS)激活的Toll样受体4(TLR4)信号对骨形态发生蛋白9(BMP9)诱导永生化小鼠胚胎成纤维细胞(i MEFs)成骨分化的影响。方法细胞免疫荧光检测TLR4/NF-κB信号通路的激活;LPS,BAY11-7082和BMP9处理iMEFs,ALP染色和活性检测i MEFs早期成骨分化能力;茜素红S染色检测晚期成骨分化能力;半定量PCR和Western blot检测晚期成骨基因OCN和OPN表达;Western blot检测Smad1/5/8磷酸化水平;半定量PCR和Western blot检测成骨关键转录因子Runx2和Dlx5的表达。结果 LPS成功激活TLR4/NF-κB信号通路;LPS抑制BMP9诱导的ALP染色和活性(P0.01)、钙盐沉积、OCN的mRNA和蛋白质表达(P0.05)、OPN的mRNA(P0.01)和蛋白质(P0.05)表达、Smad1/5/8信号通路激活(P0.01)、Runx2的mRNA和蛋白质表达(P0.05)、Dlx5的mRNA(P0.01)和蛋白质(P0.05)表达,BAY11-7082可以部分逆转LPS的抑制作用(P0.05)。结论 LPS激活TLR4可以通过NF-κB信号通路抑制BMP9诱导的iMEFs成骨分化。     

骨形态发生蛋白及其活性影响因素探讨

骨形态发生蛋白（ＢＭＰｓ）是骨和软骨诱导形成的分子调节器,它调节着骨母细胞的迁移、增殖、分化,对它的研究会加速骨组织工程的进展。研究表明,ＢＭＰｓ是一组复合体,它家族中的每一个成员以及复合体均有诱导成骨的活性,但其分子形态、浓度、所处的微环境等都影响着它的骨效应。本文简述了它的结构,并重点探讨了在骨组织工程研究中影响ＢＭＰｓ活性的因素     

骨形态发生蛋白2诱导慢性腱病大鼠肌腱干细胞体外成骨、成软骨分化

背景：目前临床对于慢性腱病缺乏有效的治疗手段,原因在于其发病机制至今尚未阐明。 目的：研究体外骨形态发生蛋白2对胶原酶诱导的大鼠慢性腱病模型髌腱来源肌腱干细胞的成骨、成软骨分化的作用。 方法：从大鼠慢性腱病模型的髌腱中分离培养出原代肌腱干细胞,传代培养至第3代细胞,行成骨、成脂、成软骨诱导分化鉴定其干细胞的特性。将肌腱干细胞(P3)单层培养至细胞融合,用重组人骨形态发生蛋白2干预。7 d后分别行茜素红染色,并行茜素红染色定量分析。将肌腱干细胞体外三维微球培养后分为2组,诱导组用重组人骨形态发生蛋白2干预,对照组不进行干预。21 d后三维微球行苏木精-伊红染色,阿利辛蓝染色以及Sox9和Ⅱ型胶原免疫组织化学染色。 结果与结论：慢性腱病大鼠来源原代肌腱干细胞体外培养呈克隆样集落生长,传代后细胞主要表现为多突的纺锤形和星形的扁平细胞,具有成纤维细胞样的特征。肌腱干细胞(P3)成脂诱导10 d,油红O染色阳性;成骨诱导7 d,茜素红染色阳性;成软骨诱导14 d,苏木精-伊红染色阳性可见软骨样细胞,Ⅱ型胶原免疫组化染色阳性。单层培养的肌腱干细胞用重组人骨形态发生蛋白2诱导7 d茜素红染色阳性,对照组为阴性,茜素红染色定量检测显示差异有显著性意义。重组人骨形态发生蛋白2诱导肌腱干细胞21 d,苏木精-伊红染色可见软骨样细胞形成、阿利辛蓝染色可见细胞内糖胺多糖沉积、Sox9和Ⅱ型胶原免疫组织化学染色均呈阳性。可见体外重组人骨形态发生蛋白2可以诱导慢性腱病来源的肌腱干细胞成骨、成软骨分化。这为进一步研究慢性腱病的发病机制提供了细胞生物学依据。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

利用重组人骨形态发生蛋白-2诱导椎间盘成骨的实验研究

目的确定在兔子的椎间盘内注射重组人骨形态发生蛋白-2（recombinant human bone morphogenetic protein-2，rhBMP-2）诱导椎体间融合的可行性。方法将24只成年新西兰大白兔，随机分为2组，每组12只。用微量注样器将含有rhBMP-2200μg的生理盐水溶液20μl和等量的生理盐水分别注射到成年新西兰大白兔的L4～5椎间盘的髓核内。术后10、30、60及90天进行X线照相和组织学检查。结果注射椎间盘未见免疫排斥反应。实验组可见纤维环和软骨终板成骨并在相邻椎体间形成骨桥。对照组的椎间盘内未见骨形成。结论利用注射的方法，rhBMP-2可诱导椎间盘成骨，达到椎体间融合的目的。     

骨形态发生蛋白2和7共转染骨髓间充质干细胞的成骨分化

背景：骨形态发生蛋白最主要的作用是诱导骨形成,但因提取困难、代谢速度快、难以准确控制其使用浓度、价格昂贵等限制了其在体外和体内相关研究中的应用。 目的：构建含特异细胞生长因子基因骨形态发生蛋白2,7的腺病毒载体,观察骨形态发生蛋白2和骨形态发生蛋白7基因共转染对兔骨髓间充质干细胞成骨分化的促进作用。 方法：将全骨髓贴壁法分离得到兔骨髓间充质干细胞传至第3代,分为5组,空白组和常规诱导组分别以常规培养基和成骨诱导分化培养基培养;骨形态发生蛋白2, 7腺病毒转染组：分别单独以骨形态发生蛋白2、骨形态发生蛋白7腺病毒进行转染;联合转染组：以2种骨形态发生蛋白腺病毒联合转染。 结果与结论：转染第7天,联合转染组成骨相关基因Runx-2、Osx、Ⅰ型胶原和碱性磷酸酶mRNA表达均较其他各组增高(P < 0.05);骨形态发生蛋白2,7腺病毒转染组上述指标较空白组和常规诱导组增高(P < 0.05)。转染第14天,联合转染组4个指标均较其他各组明显增高(P < 0.05);同时,4个指标表达均高于第7天(P < 0.05)。病毒转染后第7天,联合转染组Ⅰ型胶原和骨钙素蛋白表达均较其他组明显增高(P < 0.05)。提示对骨髓间充质干细胞进行骨形态发生蛋白2腺病毒和骨形态发生蛋白7腺病毒共转染后,较单基因转染和成骨诱导液诱导更能促进成骨相关基因和蛋白的表达。     

离子造影剂增强Micro-CT扫描对大鼠关节软骨形态的定量分析

利用Micro-CT技术的较高分辨精度,探索一种新的测量小动物关节软骨形态的方法,为进一步利用小动物模型开展关节软骨的损伤和退变等研究提供基础。选择不同浓度的造影剂及Micro-CT扫描条件,通过造影剂对比增强Micro-CT扫描大鼠膝关节,对大鼠股骨远端关节软骨进行手动分割、重建,得到软骨3D模型,利用重建得到的3D模型,对软骨的厚度和体积等形态学参数进行无创定量分析。根据造影剂、软骨和软骨下骨对X射线吸收的平均相对衰减值差异,确定30%的离子造影剂浓度为最佳。利用该浓度对4个不同的正常大鼠关节软骨进行扫描和重建,分析获得关节软骨的体积和厚度,并求得关节软骨体积和厚度的变异系数均方根分别为7.3%和6.1%。结果提示,利用造影剂增强Micro-CT扫描能够对软骨形态重要参数厚度、体积等进行定量评价。     

The vascular collar of the ilium— Three‐dimensional evaluation of the dominant nutrient foramen

Nutrient arteries are the predominant blood supply to endochondral bones and are particularly important during the early stages of endochondral ossification and the active growth period. These nutrient vessels traverse the periosteal shell of a developing bone to invade the disintegrating cartilage matrix and bring about endochondral bone formation. This results in the formation of a nutrient foramen which is retained as the vascular conduit between the exterior and interior of the bone. This study examined the dominant nutrient foramen of the neonatal ilium using high resolution micro‐computed (micro‐CT) tomography. Three‐dimensional reconstruction of micro‐CT data consistently demonstrated the presence of a distinctive, yet poorly reported, collar of bone extending into the trabecular cavity beyond the endosteum. This study proposes that this collar of bone may have formed in response to osteogenic signaling from approximated arterial vasculature. Additionally, it is suggested that the formation of this collar may act as a protective mechanism to the dominant nutrient vessel and as a potential biomechanical anchor for surrounding trabeculae, aiding to increase the biomechanical competency around the area of the foramen. The documentation of this osteological structure is important from a clinical perspective to prevent the misinterpretation of fracturing and pathology on plain plate radiographs and clinical computed tomography scans. Clin. Anat., 2013. © 2013 Wiley Periodicals, Inc.     

Ectopic Osteogenesis by Ex Vivo Gene Therapy Using Beta Tricalcium Phosphate as a Carrier

Injuries and other damage to large bone can result in defects that do not heal spontaneously and lead to severe functional impairment. Better therapies are greatly needed to address this worldwide problem. The objective of the present study was to determine whether adenoviral delivery of modified human BMP2 gene (AdBMP2) using beta tricalcium phosphate (ß-TCP) as a carrier could promote osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) and ectopic bone formation. Rabbit BMSCs were separated from tibia aspirates and expanded in vitro. The BMSCs were then infected with AdBMP-2. Expression of BMP2, alkaline phosphatase, type I collagen, osteonectin, osteopontin, and mineralization of the cells confirmed secretion of active BMP2. Cells were observed to differentiate and maintain the osteoblast phenotype. For additional in vivo experiments, subcutaneous pockets were created on the backs of nude mice, which were then implanted with AdBMP2-BMSCs/ß-TCP, Adβgal-BMSCs/ß-TCP, BMSCs/ß-TCP, or ß-TCP alone. The nude mice were sacrificed after 4 weeks for histological evaluation. Adβgal-BMSCs/ß-TCP, BMSCs/ß-TCP, and ß-TCP did not show bone formation, although extensive fibrous tissue formed in the subcutaneous space in the rats implanted with ß-TCP. However, new bone tissue formation was observed on the inner walls of the pores of the ß-TCP-treated animals, and ectopic bone formation (mainly ‘‘cartilage-bone inducing’’) was observed in the AdBMP2-BMSCs/ß-TCP composite. These results confirmed the osteogenic potential of BMSCs after AdBMP2 transduction and revealed that AdBMP2-BMSC/ß-TCP composites could provide the capacity for bone formation and maturation during the more advanced stages of healing.     

Osteoinduction of porous Ti implants with a channel structure fabricated by selective laser melting

Many studies have shown that certain biomaterials with specific porous structures can induce bone formation in non-osseous sites without the need for osteoinductive biomolecules, however, the mechanisms responsible for this phenomenon (intrinsic osteoinduction of biomaterials) remain unclear. In particular, to our knowledge the type of pore structure suitable for osteoinduction has not been reported in detail. In the present study we investigated the effects of interconnective pore size on osteoinductivity and the bone formation processes during osteoinduction. Selective laser melting was employed to fabricate porous Ti implants (diameter 3.3mm, length 15 mm) with a channel structure comprising four longitudinal square channels, representing pores, of different diagonal widths, 500, 600, 900, and 1200 μm (termed p500, p600, p900, and p1200, respectively). These were then subjected to chemical and heat treatments to induce bioactivity. Significant osteoinduction was observed in p500 and p600, with the highest observed osteoinduction occurring at 5mm from the end of the implants. A distance of 5mm probably provides a favorable balance between blood circulation and fluid movement. Thus, the simple architecture of the implants allowed effective investigation of the influence of the interconnective pore size on osteoinduction, as well as the relationship between bone quantity and its location for different pore sizes.     

Assessment of Typhoid Vaccines by Using the Intraperitoneal Route of Challenge

Present laboratory tests for human typhoid vaccines use an intraperitoneal route of challenge given 7 days after injection of increasing doses of standard and test vaccines by the same route. In studies reported here, groups of B6D2 mice were vaccinated intraperitoneally with 2 x 10(8) acetone-killed Salmonella typhi Ty2, with the Vi antigen-free variant O-901, or with Yersinia enterocolitica and Serratia marcescens suspensions. Other groups of mice received 200 mug of purified S. typhi or S. marcescens endotoxin, or their corresponding purified lipid A components. All of the vaccinated mice (except for saline- or thioglycolate-injected controls) exhibited increased protection against the lethal intraperitoneal challenge with S. typhi Ty2. Serial quantitative bacterial counts carried out on peritoneal washouts and on homogenates of the draining mediastinal lymph nodes indicated the development of an antibacterial response by the vaccinated host which was not observed in the control animals. Mice receiving purified endotoxin (lipopolysaccharide) exhibited varying degrees of protection, both in terms of increased host survival and the amount of inactivation of the challenge population in vivo. The response seen when the antigenically unrelated S. marcescens lipopolysaccharide was injected was little different from that seen when the acetone-killed S. typhi Ty2 whole-cell vaccine was used. This suggests that nonspecific inactivation of the intraperitoneal challenge contributes substantially to the immune response seen in mice vaccinated intraperitoneally with specific typhoid antigens.     

异位嗜铬细胞瘤的CT诊断价值

目的探讨异位嗜铬细胞瘤的CT诊断价值,指导临床手术,降低手术风险。方法回顾性分析10例异位嗜铬细胞瘤的临床资料及CT表现,并复习文献。结果10例异位嗜铬细胞瘤术前均进行CT平扫及增强检查,8例位于腹主动脉旁,1例位于膀胱,1例位于后纵隔。平扫肿瘤呈类圆形或椭圆形,伴有囊变、坏死及钙化密度常不均匀;动脉期明显强化,病灶内或周围见增粗迂曲血管影;实质期病灶强化程度稍下降或持续强化。结论 CT检查对异位嗜铬细胞瘤的定位、定性有重要价值,并对手术方案制定及术后的随访有重要意义。