Name: computed tomography and optical imaging of osteogenesis-angiogenesis coupling to assess integration of cranial bone autografts and allografts
Uploaded: 2015-12-22T13:00:00.000+00:00
Duration: 13 min 16 s
Description: Watch this Scientific Journal Video about Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts at JoVE.com

The authors acknowledge funding from the NIDCR (Grant No. DE019902) and from the Israeli Science Foundation (Grant No. 382/13).

该协议遵循耶路撒冷，以色列（申请号MD-12-13524-4），经批准AAALAC设施和由雪松 - 西奈医学中心的希伯来大学的体制动物护理和使用委员会（IACUC）的指导方针IACUC（申请号3770）。这些动物是在严格遵守美国国立卫生研究院的指导方针进行处理。 1.准备骨移植物<ol><li>安乐死7〜8周龄的Balb / C小鼠，或任何应变来自受体不同，使用的CO 2的吸入或腹腔注射50微升苯巴比妥（65毫克/毫升; 100毫克/千克）的标准方法。确认没有心跳并执行颈椎脱位。可替代地，从已被保存在-20℃，收获前解冻屠体收获了同种异体移植。 </li><li>用理发推子，把它应用逆着毛发生长的方向剃须颅骨区域。通过将70％消毒尸体头部isopropano湖使用11号手术刀切开头皮及去除骨膜。 </li><li>用牙钻和4.5毫米内径的环钻，分离颅骨的圆形片段。转移骨片段含有无菌盐水的100毫米的塑料培养皿中。此时，观察血并附着到骨片段（图1A）的软组织。从22供体获得骨碎片以这种方式。 </li><li>容纳一个骨碎片，在采用弧形镊子时间。擦拭它彻底用棉签取出所有的脑组织，软组织和血液（ 图1B）。轻轻地切掉了保持连接到用细剪的移植，使移植物将有一个完美的圆形任何碎骨片。 </li><li>在一个15毫升管含有70％的乙醇和含磷酸盐缓冲盐水洗两次乙醇在15ml试管浸各接枝一次。 </li><li>冻结在-80℃的冰箱中的移植在冷冻管在至少1周。在这一点上，确保移植是透明（ 图1C）。 </li></ol> 2.颅骨缺损手术<ol><li>对于收件人使用的表达骨钙素子6控制下的荧光素酶的转基因小鼠。 </li><li>消毒的使用玻璃珠加热器30秒的外科手术工具的末端。转移到含有70％的异丙醇保持无菌条件罐子的工具。使用无菌手套。 </li><li>通过腹膜内注射氯胺酮一个美托咪定的混合物（75毫克/公斤和1毫克/千克，分别）的麻醉一个7〜8周龄的雌性小鼠。捏动物的肢体，以确保其深度麻醉。不要让动物无人看管，直到它重新获得足够的意识，以保持胸骨斜卧。 </li><li>通过将它逆着毛发生长的方向剃须用理发推子的颅骨区域。适用于毛发去除霜，以消除任何残余的皮毛和擦拭AFTEř使用干纱布随后盐水浸泡的纱布不超过2分钟。应小心，以避免在眼睛上。另外，兽医眼膏可先脱毛应用为保护眼睛额外的一层。当务之急是的脱毛霜所有痕迹，以避免过度暴露于化学试剂可能刺激被除去。通过将5％的氯己定（图1D）消毒头皮。 </li><li>注入皮下5mg / kg的卡布洛芬稀释在200μl盐水中。应用兽医眼药膏，以防止干燥和保持动物在任何时候一个散热垫。定位在操作望远镜的动物。 </li><li>请用细剪刀和镊子（ 图1E）1厘米长的椭圆形切割的头皮。按住采用弧形镊子骨膜切除用细剪刀。 </li><li>用牙钻钻颅骨缺损2毫米前到人字缝第二一块5mm外径环钻（图1F 和G）。为了避免硬脑膜渗透，钻四分之三的方式进入骨和使用刮刀分离骨片段。 </li><li>植入自体，获取的骨头碎片和含有10ml无菌盐水，清除杂物10毫米塑料板清洗。不要取出软组织。植入同种异体移植物，解冻提前​​同种异体移植物，并将其标准化至室温，然后再清洗。 </li><li>放置植骨采用弧形镊子，所以它不会触及缺陷利润率（ 图1H）的缺陷。胶接枝使用纤维蛋白胶宿主骨（5微升46毫克/毫升纤维蛋白原，用5微升25单位/毫升凝血酶混合）。 </li><li>缝合使用4-0薇乔缝合（图1I）的皮肤。应用聚维酮碘的手术切口。应注意，以防止缝合线的污染毛皮。马克小鼠耳，注入0.25毫克/千克Antisedan牛逼Ø扭转美托咪定的麻醉效果。 </li><li>如果动物没有在10分钟内醒来，确保它坐落在一个温暖垫。注入200μl的无菌盐水，并在需要时注射0.1毫克/公斤Antisedan。不返回已经历手术给其他动物的公司，直到完全恢复的动物。 </li><li>请分开的笼中的动物了一个星期，以防止他们打开缝线。放置小鼠食物在水中浸泡在培养皿上的箱地板几天术后。注入皮下200微升手术来治疗手术后疼痛后在无菌盐水每天一次稀释3天1mg / ml的溶液卡洛芬。 </li></ol><img alt="图1" src="/files/ftp_upload/53459/53459fig1.jpg" /> 图1.颅骨同种异体制备和颅骨缺损外科的骨片段从颅骨区域 （A）中分离。软组织，骨髓和血液被擦拭（B）和接枝漂洗在70％乙醇，随后通过洗涤两次在磷酸盐缓冲盐水（C，AG =同种异体移植物）。收件人鼠标在颅盖区域（D）的手术用剃须和皮肤消毒制备。一个椭圆形的切口在头皮皮肤创建，骨膜被除去（E）。接着，颅骨缺损是使用5毫米直径的环钻（F）的钻孔。骨片段是使用勺形刮刀拆下，留下上矢状窦完好（G）。同种异体移植物，然后放置在缺陷，并使用纤维蛋白胶（H）的固定。最后，皮肤缝合（ 我 ）。 <a href="https://www.jove.com/files/ftp_upload/53459/53459fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.微计算机断层扫描的表征血管树<ol><li>准备肝素生理盐水。重量2000ü肝素钠，并将其放置于50ml的塑料管。加入20 5ml盐水，摇匀管。填有20毫升的Luer锁注射器用15ml温热肝素生理盐水和注射器连接到一个23-G的头皮静脉集。加盖注射器到注射器泵，并将其设置为泵10混合物在1毫升/分钟。 </li><li>稳重一个使用腹膜内注射50μl的苯巴比妥（65毫克/毫升; 100毫克/千克）的小鼠。捏动物的肢体，以确保它深深地镇静剂。固定鼠标在它的后面，以一个固定板包裹有吸收性表面衬套（ 图2A）。将鼠标固定在通风橱中，以防止人员暴露在烟气。 </li><li>切从腹部到使用细剪刀和镊子（图2B）的颈部皮肤。切开腹壁到剑突（图2C）。切割沿其横向方面的肋笼。避免伤及肺部的ð心脏，并确保心脏完全暴露。使用止血钳缩回胸骨（图2D）。 </li><li>插入蝴蝶针3毫米到左心室（图2E）。胶针用3秒胶（ 图2F）的心脏和胸壁。 </li><li>立即启动泵。一分钟后，解剖下腔静脉，以减压的血管（ 图2G）。倾斜板，使液体将流入远离头部。成功的灌注将导致清除肝脏和血管。 </li><li>当肝素盐水灌注完成，灌注10毫升4％的甲醛。避免气泡渗入脉管并避免接触到的甲醛气体。成功的甲醛灌注会导致加劲的尾巴。冲洗用肝素化食盐水10ml甲醛。 </li><li>根据厂家专业制备不透射线的造影剂urer说明。通常情况下，这将需要的化合物，稀释剂和固化剂的混合。使用血清移液管，混合在15毫升塑料管的解决方案。 </li><li>以1毫升/分钟的速率灌注与造影剂的混合物的动物。观察冠状动脉，肠和肝脏的血管，并确保他们是枯黄的2灌流后- 3毫升，表示成功灌注（ 图2H）。当灌注完成后，切蝴蝶针的管子，独自一人获得了尸体，并用卫生纸包起来，以防止解决了颅骨区域的泄漏。允许聚合在4°CO / N。 </li><li>解剖颅骨区域（ 图2I）;首先用手术刀切开皮肤横向越好，避免损伤感兴趣的颅地区。找到骨移植，然后用细剪刀剪开颅骨骨10 mm处的移植。 </li><li>扫描隔离CALV使用μCT扫描仪宋体骨骼样本;设定的视野到20.5毫米，55 kVp的X射线能量，145微安强度，使用每360°和积分时间的200毫秒1000突起。调整μCT参数7图像其他骨缺损模型。 </li><li>观察由μCT软件中生成的2D重建的切片。确保扫描感兴趣的区域是正确的，然后找到了颅骨缺损。评估灌注的质量。成功鉴定血管（图2J）后，继续和脱钙使用6％三氯乙酸（TCA）稀释在双蒸水（DDW）的样品。 </li><li>重新扫描使用第3.10节所示的参数样本。定位所重建的二维切片的人字缝。定义在2mm和直径8mm高度的兴趣（VOI）的圆柱形体积，即正切于缝合 - 这是缺陷的解剖部位。 </li><li>段次鄂骨组织使用一个全局阈值方法软组织;较低的门槛设置为130，并应用约束的3D高斯滤波器（SIGMA] [σ= 2.0，并支持= 2）部分的体积抑制噪声。生成一个三维重建，并确保VOI正确定位（ 图2K）。 </li><li>生成使用IPL软件和"dt_object_param" 功能 （图2L）的厚度地图。 注:该函数描述了容器体积为每个血管直径。 </li></ol><img alt="图2" src="/files/ftp_upload/53459/53459fig2.jpg" /> 图2.通过灌注心脏的方法使用的是不透射线剂，鼠标被固定到固定板（A）中，与皮肤沿腹部到颈部（B）的切断。腹壁沿着内侧排队至剑突PROCE切分类（C），以及胸廓横向（D）的切割。甲蝶针插入左心室，避免插入右心室，这是明显更暗（E中，右心室是由白虚线表示），并且针固定到心脏和胸腔壁（F）的 。肝素化的盐水（2-3毫升）泵入到心脏，并且下腔静脉解剖（G，白色箭头表示下腔静脉）。甲醛（4％），灌注和冲洗掉;这之后是黄染无线电不透明造影剂的灌注。成功灌注将明显受血管，从而改变颜色，黄色（H）的间隙。聚合后，颅盖区域分离（Ⅰ，AG =同种异体移植物），并用μCT扫描器，以确认正确的灌注（J，感兴趣卷被用橙色）成像。该样品脱钙和重新扫描（K），接着是生成的彩色厚度图（L）。 <a href="https://www.jove.com/files/ftp_upload/53459/53459fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 骨矿化和血液灌注4.荧光成像<ol><li>重构探针的2纳摩尔/ 100微升的PBS在1.5ml管中的浓度（使用二膦酸 - 共轭荧光探针来监测骨矿化和血源性荧光剂来测量血液灌注）。吸管轻轻几次，以确保探针的均质溶解。 </li><li>指定的成像会议之前24小时取一次零的图像;麻醉用2升/分​​钟吸入100％医疗级氧的感应腔室补充有3％异氟醚的3只小鼠。经过适当的麻醉已经建立，降低异氟烷浓度为1.5％ - 2％。 </li><li>应用兽医眼药膏，以防止干燥，并确保散热垫接通时刻。捏动物的肢体，以确保其深度麻醉。不要让动物无人看管，直到它重新获得足够的意识，以保持胸骨斜卧。 </li><li>用理发推子应用它逆着毛发生长的方向剃须颅骨区域。删除使用毛发去除霜毛皮残余。任何皮草残留会干扰光学成像。取出用细剪刀和镊子缝线。 </li><li>放置三只动物在IVIS阶段加热到37℃，每个成像会话。置的曝光时间，以自动，查看字段至C，并调整过滤器。 例如，对于二膦酸-共轭荧光探针，其特征与最大激发在680纳米的光波长，使用675纳米的激发滤光片和经Cy5.5的发射滤波器。同样地，使用的是血源性荧光剂，峰值激发时在光波长750nm的使用的710纳米的激发滤光片和780nm的发射滤光片。 </li><li>图像通过按"获取"按钮生活图象软件的小鼠。确保颅骨区域完全可见（ 图3B）对于一个全面的指南劝廉等人 2009年8月。 </li><li>让老鼠清醒吸入100％的氧气。 </li><li>通过静脉注射尾注入2纳摩尔荧光探针。在指定的成像会议，如上所述进行成像。 </li></ol> 5.微型计算机断层扫描用于骨骼再生<ol><li>用于骨形成的定量，麻醉小鼠通过吸入3％异氟烷的，如在步骤4.2中描绘 - 4.3。传输鼠标的μCT扫描仪床上，并将其放置在扫描仪上。 </li><li>设置μCT扫描器的x射线管势能到55 kVp的和为145微安中等分辨率强度。使用的视图20.5 mm的场中，执行侦察员（单个X射线）扫描和定义从鼠标的眼睛延伸到它的头骨的背面感兴趣的区域。进行CT扫描，使用每200毫秒360°和积分时间1000的预测。 </li><li>重建使用20μm的标称空间分辨率的二维切片。对齐缺陷利润率使用μCT软件9标准化的位置。 </li><li>类似于步骤3.20中，定义感兴趣的圆柱形体积并应用约束三维高斯滤波器（σ= 0.8和支持= 1），以部分地抑制在卷噪声。段从利用全局阈值的过程的软组织的骨组织。 </li><li>确定矿化骨组织在感兴趣体积的体积。 </li></ol>干细胞招募和分化6.生物发光成像<ol><li>准备鼠标成像所描绘的第4.2-4.4和清醒小鼠吸入100％的氧气。 </li><li>辖126毫克/千克甲虫荧光素经由腹膜内注射各小鼠，并返回动物给感应腔室。 2分钟后，麻醉用3％异氟烷的动物。放置在IVIS小鼠温热阶段。 </li><li>通过活影像软件，将曝光时间设置为"自动"，并且视场为C.图片荧光素给药后小鼠5分钟。图像如在步骤4.6中描述的动物，使用"生物发光"模态定义感兴趣的区域，因为小鼠之间的转基因表达变化。规范化颅骨信号到尾。 </li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTH-enhanced+structural+allograft+healing+is+associated+with+decreased+angiopoietin-2-mediated+arteriogenesis,+mast+cell+accumulation,+and+fibrosis.">PTH-enhanced structural allograft healing is associated with decreased angiopoietin-2-mediated arteriogenesis, mast cell accumulation, and fibrosis.</a> J Bone Miner Res. 28 (3), 586-597 (2013).</li><li>Nebuloni, L., Kuhn, G. A., Vogel, J., Muller, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vivo+vascular+imaging+approach+for+hierarchical+quantification+of+vasculature+using+contrast+enhanced+micro-computed+tomography.">A novel in vivo vascular imaging approach for hierarchical quantification of vasculature using contrast enhanced micro-computed tomography.</a> PLoS One. 9 (1), e86562 (2014).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periosteal+progenitor+cell+fate+in+segmental+cortical+bone+graft+transplantations:+implications+for+functional+tissue+engineering.">Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.</a> J Bone Miner Res. 20 (12), 2124-2137 (2005).</li><li>Movahed, R., Pinto, L. P., Morales-Ryan, C., Allen, W. R., Wolford, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+cranial+bone+grafts+for+reconstruction+of+maxillofacial+deformities.">Application of cranial bone grafts for reconstruction of maxillofacial deformities.</a> Proc (Bayl Univ Med Cent). 26 (3), 252-255 (2013).</li><li>Putters, T. F., Schortinghuis, J., Vissink, A., Raghoebar, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+study+on+the+morbidity+resulting+from+calvarial+bone+harvesting+for+intraoral+reconstruction.">A prospective study on the morbidity resulting from calvarial bone harvesting for intraoral reconstruction.</a> Int J Oral Maxillofac Surg. , (2015).</li><li>Kline, R. M., Wolfe, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+associated+with+the+harvesting+of+cranial+bone+grafts.">Complications associated with the harvesting of cranial bone grafts.</a> Plast Reconstr Surg. 95 (1), 5-13 (1995).</li><li>Hassanein, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+calvarial+burring+on+resorption+of+onlay+cranial+bone+graft.">Effect of calvarial burring on resorption of onlay cranial bone graft.</a> J Craniofac Surg. 23 (5), 1495-1498 (2012).</li><li>Yin, J., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Completely+resorption+of+autologous+skull+flap+after+orthotopic+transplantation:+a+case+report.">Completely resorption of autologous skull flap after orthotopic transplantation: a case report.</a> Int J Clin Exp Med. 7 (4), 1169-1171 (2014).</li><li>Schuss, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+flap+resorption:+risk+factors+for+the+development+of+a+long-term+complication+following+cranioplasty+after+decompressive+craniectomy.">Bone flap resorption: risk factors for the development of a long-term complication following cranioplasty after decompressive craniectomy.</a> J Neurotrauma. 30 (2), 91-95 (2013).</li><li>Ben Arav, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-coated+allografts:+a+novel+approach+for+cranioplasty.">Adeno-associated virus-coated allografts: a novel approach for cranioplasty.</a> J Tissue Eng Regen Med. 6 (10), e43-e50 (2012).</li><li>Ito, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+cortical+bone+allografts+mediated+by+adherent+rAAV-RANKL+and+VEGF+gene+therapy.">Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy.</a> Nat Med. 11 (3), 291-297 (2005).</li><li>Sheyn, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTH+promotes+allograft+integration+in+a+calvarial+bone+defect.">PTH promotes allograft integration in a calvarial bone defect.</a> Mol Pharm. 10 (12), 4462-4471 (2013).</li><li>Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+vessel+maturation.">Molecular regulation of vessel maturation.</a> Nat Med. 9 (6), 685-693 (2003).</li><li>Reginato, S., Gianni-Barrera, R., Banfi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taming+of+the+wild+vessel:+promoting+vessel+stabilization+for+safe+therapeutic+angiogenesis.">Taming of the wild vessel: promoting vessel stabilization for safe therapeutic angiogenesis.</a> Biochem Soc Trans. 39 (6), 1654-1658 (2011).</li><li>Moutsatsos, I. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenously+regulated+stem+cell-mediated+gene+therapy+for+bone+regeneration.">Exogenously regulated stem cell-mediated gene therapy for bone regeneration.</a> Mol Ther. 3 (4), 449-461 (2001).</li><li>Deckers, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+proteins+stimulate+angiogenesis+through+osteoblast-derived+vascular+endothelial+growth+factor.">Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor.</a> A. Endocrinology. 143 (4), 1545-1553 (2002).</li><li>Cornejo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+adipose+tissue-derived+osteogenic+and+endothelial+cells+on+bone+allograft+osteogenesis+and+vascularization+in+critical-sized+calvarial+defects.">Effect of adipose tissue-derived osteogenic and endothelial cells on bone allograft osteogenesis and vascularization in critical-sized calvarial defects.</a> Tissue Eng Part A. 18 (15-16), 1552-1561 (2012).</li></ol>

The authors have nothing to disclose.

这里描述的多模成像方法的目的是为了使血管发生成骨轴的颅骨移植的上下文细致的调查。使用μCT协议，这使得血管树的准确高分辨率三维演示喂养整个颅骨缺损新生血管成像。 μCT数据可以使用先进的工具，如IPL的软件很容易分析。例如， 在图3C中示出的厚度的分析显示，自体颅和同种异体移植物之间的主要差别是在容器为0.1至0.08毫米的直径，其表征血液动脉10。这一结果...

颅面骨量丢失，由于外伤，肿瘤切除，开颅减压术，先天性缺陷很少治愈本身，并提出了明确的未满足的临床需求。自体骨移植物和同种异体骨移植物被广泛地用于治疗这些病症1。 它已被广泛接受的成骨紧密结合与血管生成2,3。因此，提出了治疗骨再生的完整研究应包括血管树整个缺损部位形成的一个全面的调查。有几种可用的方法研究模型来刻画血管。血管树可以通过组织学分析进行调查。自组织学依赖于切片的组织，存在所得到的图像将被扭曲的可能性高。为了解决这个问题，活体显微镜可以执行将图像的完好的血管4;然而，这种方法是限制在一个平面上成像。从灌注造影剂的动物获取的样品μCT扫描允许饲料再生网站5血管网络的3D影像。这种方法允许器官的脉管作为一个整体的一个非常详细的示范，以及血管分布的细致的分析。此外，μCT使多样直径血管，这表征血管的不同亚型之间的分化。 我们假设一个颅骨自体的该植入会诱发比同种异体移植物的植入更大新血管形成，而这增加了新血管形成将导致反过来增强骨formation.To追求这一假设，我们所采用的各种技术。我们通过执行μCT为基础的分析研究了新成立的血管树的模式。我们用血池荧光探针测量血液灌注。接下来，我们的驴SED骨组织矿化羟基磷灰石定向探头和μCT分析FLI。最后，我们监测干细胞招募和分化，在将荧光素酶表达在骨钙素阳性细胞的转基因小鼠进行的BLI。

<table><tbody><tr><td>C57BL/C&amp;nbsp; Mice</td><td>Harlan laboratories</td><td>57</td><td></td></tr><tr><td>FVB/n Mice</td><td>Harlan laboratories</td><td>862</td><td></td></tr><tr><td>Phenobarbital</td><td>West waro</td><td>NDC 0641-0477-25</td><td></td></tr><tr><td>Rodent hair clipper</td><td>Wahl animal</td><td>8786-451A</td><td></td></tr><tr><td>Scalpel 11</td><td>Miltex</td><td>27111504</td><td></td></tr><tr><td>Dental micro motor</td><td>marathon III</td><td></td><td></td></tr><tr><td>5 mm trephine</td><td>Fine Science tools</td><td>18004-50</td><td></td></tr><tr><td>Hair removing cream</td><td>Veet</td><td></td><td></td></tr><tr><td>KetaVed (Ketamine)</td><td>Vedco</td><td>NDC 50989-996-06</td><td></td></tr><tr><td>Domitor</td><td>Zoetis</td><td>NADA 141-267</td><td></td></tr><tr><td>carprofen</td><td>Norbrook</td><td>02000/4229</td><td></td></tr><tr><td>Eye ointment</td><td>Puralube</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Operating binocular</td><td>Kent scientific</td><td>KSCXTS-1121</td><td></td></tr><tr><td>Fine scissors&amp;nbsp;</td><td>Fine Science tools</td><td>14060-11</td><td></td></tr><tr><td>Curve tweezers</td><td>Fine Science tools</td><td>11274-20</td><td></td></tr><tr><td>Spoon shaped spatula</td><td>Fine Science tools</td><td>10090-13</td><td></td></tr><tr><td>Tisseel Fibin gel kit&amp;nbsp;</td><td>Baxter</td><td>718971</td><td></td></tr><tr><td>needle holder</td><td>Fine Science tools</td><td>12060-01</td><td></td></tr><tr><td>vicryl suture 4-0</td><td>Ethicon</td><td>J392H</td><td></td></tr><tr><td>Antisedan</td><td>Zoetis</td><td>NADA#141033</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>20 ml luerlock&amp;nbsp;</td><td>BD</td><td>302830</td><td></td></tr><tr><td>23G scalp vein set (butterfly needle)</td><td>BD</td><td>367342</td><td></td></tr><tr><td>Hemostat</td><td>Fine Science tools</td><td>13008-12</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard apparatus</td><td>PHD 2000</td><td></td></tr><tr><td>3-sec gel glue&amp;nbsp;</td><td>Scotch</td><td></td><td></td></tr><tr><td>rodent dissection board</td><td>Leica</td><td>38DI02313</td><td></td></tr><tr><td>Microfil MV-122</td><td>flow-tech</td><td>MV-122</td><td></td></tr><tr><td>&amp;#956;CT40 scanner</td><td>Scanco</td><td>&amp;#956;CT40</td><td></td></tr><tr><td>TCA6%</td><td>Sigma</td><td>T6399</td><td></td></tr><tr><td>Osteosense 680</td><td>PerkinElmar</td><td>NEV10020EX</td><td></td></tr><tr><td>Angiosense750</td><td>PerkinElmar</td><td>NEV10011</td><td></td></tr><tr><td>Oxigen 100% medical grade</td><td></td><td></td><td></td></tr><tr><td>isoflurane (furane)</td><td>Baxter</td><td>1001936040</td><td></td></tr><tr><td>IVIS kinetics</td><td>Xenogen</td><td></td><td></td></tr><tr><td>Beetle luciferin</td><td>Promega</td><td>E160A</td><td></td></tr></tbody></table>

computed tomography and optical imaging of osteogenesis-angiogenesis coupling to assess integration of cranial bone autografts and allografts

一个主要参数确定一个骨移植过程的成功是包围接枝的区域的血管形成。我们假设一个植骨的是植入会诱发更大的骨再生的丰富的血管形成。调查接枝的对新血管形成在缺损部位的影响，我们开发了一种显微计算机断层摄影术（μCT）的方式来表征新形成的血管，其包括具有聚合的造影剂的动物的全身灌流。该方法，能够将其全部器官的血管详细分析。另外，血液灌注是使用血源性荧光剂的荧光成像（FLI）评估。骨的形成是使用羟基磷灰石针对性的探头和μCT分析FLI量化。干细胞募集是由转基因小鼠的生物发光成像（BLI），该表达骨钙蛋白启动子控制下的萤光素酶进行监测。这里，我们描述和展示制备的同种异体移植物的，颅骨缺损手术，对于新血管形成的研究和骨形成分析（包括体内灌注造影剂），并且协议进行数据分析的μCT扫描协议。 脉管系统的三维高分辨率分析表明在动物中植入自体移植显著更大的血管生成，特别是相对于小动脉的形成。因此，血液灌流是由第 7天手术后显著高于自体移植小组。我们在接受自体移植动物中观察到优越的骨矿化和测量更大的骨形成。自体移植诱导驻留干细胞募集到移植物宿主骨缝线，其中所述细胞分化成的第 7和第10日术后天之间骨形成细胞。这一发现意味着，增强骨形成可能是由于扩充血管饲养的特点自体植入。该方法的描述可作为研究骨组织再生紧密界骨骼的形成和新生血管方面的最佳工具。

新血管形成通过μCT体积分析和通过FLI使用荧光血源性剂量化血液灌注进行评估。手术后七天，μCT扫描证实在已经收到自体比在已经收到移植从C57BL / 6（图3A）收获小鼠小鼠显著更高体积的中小直径的血管。有趣的是，移植组中新形成的血管树似乎达到整个缺陷区域，而同种异体移植组中的血管出现穿透从外边缘缺损部位和向内延伸到仅在有限的程度。所述FLI支持了这一观察，示出显?...

Watch this Scientific Journal Video about Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts at JoVE.com

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

自体和异体骨移植物植入构成接受的方法来治疗严重颅面骨量丢失。然而接枝组合物对新血管形成，细胞分化和骨形成之间的相互作用的影响还不清楚。我们提出的目的是阐明血管生成骨形成相互依存的移植接近一个多模式成像协议。

计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

computed-tomography-optical-imaging-osteogenesis-angiogenesis

Research

JoVE Journal

Bioengineering

11.4K 次观看.  The Hebrew University–Hadassah Faculty of Dental Medicine. 自体和异体骨移植物植入构成接受的方法来治疗严重颅面骨量丢失。然而接枝组合物对新血管形成，细胞分化和骨形成之间的相互作用的影响还不清楚。我们提出的目的是阐明血管生成骨形成相互依存的移植接近一个多模式成像协议。 

视频: Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

科研

癌症研究

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

稀疏的头骨皮层的窗口技术<em在体内</em光学相干断层成像

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

神经科学

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

混合μCT-FMT成像和图像分析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

生物工程

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

结合<em&gt;在体内</em&gt;光学及μCT成像监视感染，炎症和骨解剖在骨科植入物感染的小鼠

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

医学

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

用 Imagej 插件对小梁骨的外边界辅助分割和量化

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

猪胫骨移植术后血管生成的动物模型

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Digital Hybrid Model Preparation for Virtual Planning of Reconstructive Dentoalveolar Surgical Procedures

Virtual, hybrid three-dimensional (3D) model acquisition is presented in this article, utilizing the sequence of radiographic image segmentation, spatial registration, and free-form surface modeling. Firstly cone-beam computed tomography datasets were reconstructed with a semi-automatic segmentation method. Alveolar bone and teeth are separated into different segments, allowing 3D morphology, and localization of periodontal intrabony defects to be assessed. The severity, extent, and morphology of acute and chronic alveolar ridge defects are validated concerning adjacent teeth. On virtual complex tissue models, positions of dental implants can be planned in 3D. Utilizing spatial registration of IOS and CBCT data and subsequent free-form surface modeling, realistic 3D hybrid models can be acquired, visualizing alveolar bone, teeth, and soft tissues. With the superimposition of IOS and CBCT soft tissue, thickness above the edentulous ridge can be assessed about the underlying bone dimensions; therefore, flap design and surgical flap management can be determined, and occasional complications may be avoided.

A workflow for creating three-dimensional (3D) virtual hybrid models has been designed based on cone-beam computed tomography dataset and intraoral optical scans utilizing radiographic image segmentation methods and free-form surface modeling. Digital models are used for the virtual planning of reconstructive dentoalveolar surgical procedures.

用于重建牙槽外科手术虚拟规划的数字混合模型制备

Virtual, hybrid three-dimensional (3D) model acquisition is presented in this article, utilizing the sequence of radiographic image segmentation, spatial ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

同步加速器微CT高通量皮质骨样品采购和分析的剖面、科林和图像处理指南

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...