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>

<ol>
	<li>Finkemeier, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-grafting+and+bone-graft+substitutes.">Bone-grafting and bone-graft substitutes.</a> J Bone Joint Surg Am. 84-A (3), 454-464 (2002).</li><li>Kanczler, J. M., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+and+angiogenesis:+the+potential+for+engineering+bone.">Osteogenesis and angiogenesis: the potential for engineering bone.</a> Eur Cell Mater. 15, 100-114 (2008).</li><li>Schipani, E., Maes, C., Carmeliet, G., Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+osteogenesis-angiogenesis+coupling+by+HIFs+and+VEGF.">Regulation of osteogenesis-angiogenesis coupling by HIFs and VEGF.</a> J Bone Miner Res. 24 (8), 1347-1353 (2009).</li><li>Huang, C., 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=Spatiotemporal+Analyses+of+Osteogenesis+and+Angiogenesis+via+Intravital+Imaging+in+Cranial+Bone+Defect.">Spatiotemporal Analyses of Osteogenesis and Angiogenesis via Intravital Imaging in Cranial Bone Defect.</a> J Bone Miner Res. , (2015).</li><li>Kimelman-Bleich, N., 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=The+use+of+a+synthetic+oxygen+carrier-enriched+hydrogel+to+enhance+mesenchymal+stem+cell-based+bone+formation+in+vivo.">The use of a synthetic oxygen carrier-enriched hydrogel to enhance mesenchymal stem cell-based bone formation in vivo.</a> Biomaterials. 30 (27), 4639-4648 (2009).</li><li>Iris, B., 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=Molecular+imaging+of+the+skeleton:+quantitative+real-time+bioluminescence+monitoring+gene+expression+in+bone+repair+and+development.">Molecular imaging of the skeleton: quantitative real-time bioluminescence monitoring gene expression in bone repair and development.</a> J Bone Miner Res. 18 (3), 570-578 (2003).</li><li>Bouxsein, M. L., 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=Guidelines+for+assessment+of+bone+microstructure+in+rodents+using+micro-computed+tomography.">Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.</a> J Bone Miner Res. 25 (7), 1468-1486 (2010).</li><li>Lim, E., Modi, K. D., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescent+imaging+of+mammary+tumors+using+IVIS+spectrum.">In vivo bioluminescent imaging of mammary tumors using IVIS spectrum.</a> J Vis Exp. (26), (2009).</li><li>Kallai, I., 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=Microcomputed+tomography-based+structural+analysis+of+various+bone+tissue+regeneration+models.">Microcomputed tomography-based structural analysis of various bone tissue regeneration models.</a> Nat Protoc. 6 (1), 105-110 (2011).</li><li>Fleming, J. T., 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+blood+flow+and+vascular+reactivity.">Bone blood flow and vascular reactivity.</a> Cells Tissues Organs. 169 (3), 279-284 (2001).</li><li>Dhillon, R. 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 border="0" cellpadding="0" cellspacing="0" width="614">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57BL/C&#160; Mice</td>
			<td style="width:112px;">Harlan laboratories</td>
			<td align="right" style="width:119px;">57</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">FVB/n Mice</td>
			<td style="width:112px;">Harlan laboratories</td>
			<td align="right" style="width:119px;">862</td>
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			<td height="42" style="height:42px;width:216px;">Phenobarbital</td>
			<td style="width:112px;">West waro</td>
			<td style="width:119px;">NDC 0641-0477-25</td>
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			<td height="21" style="height:21px;width:216px;">Rodent hair clipper</td>
			<td style="width:112px;">Wahl animal</td>
			<td style="width:119px;">8786-451A</td>
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			<td height="21" style="height:21px;width:216px;">Scalpel 11</td>
			<td style="width:112px;">Miltex</td>
			<td align="right" style="width:119px;">27111504</td>
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			<td height="21" style="height:21px;width:216px;">Dental micro motor</td>
			<td style="width:112px;">marathon III</td>
			<td style="width:119px;"></td>
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			<td height="42" style="height:42px;width:216px;">5mm trephine</td>
			<td style="width:112px;">Fine Science tools</td>
			<td style="width:119px;">18004-50</td>
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			<td height="21" style="height:21px;width:216px;">Hair removing cream</td>
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			<td height="42" style="height:42px;width:216px;">KetaVed (Ketamine)</td>
			<td style="width:112px;">Vedco</td>
			<td style="width:119px;">NDC 50989-996-06</td>
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			<td height="21" style="height:21px;width:216px;">Domitor</td>
			<td style="width:112px;">Zoetis</td>
			<td style="width:119px;">NADA 141-267</td>
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			<td height="21" style="height:21px;width:216px;">carprofen</td>
			<td style="width:112px;">Norbrook</td>
			<td style="width:119px;">02000/4229</td>
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			<td height="42" style="height:42px;width:216px;">Eye ointment</td>
			<td style="width:112px;">Puralube</td>
			<td style="width:119px;">NDC 17033-211-38</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Operating binocular</td>
			<td style="width:112px;">Kent scientific</td>
			<td style="width:119px;">KSCXTS-1121</td>
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			<td height="42" style="height:42px;width:216px;">Fine scissors&#160;</td>
			<td style="width:112px;">Fine Science tools</td>
			<td style="width:119px;">14060-11</td>
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			<td height="42" style="height:42px;width:216px;">Curve tweezers</td>
			<td style="width:112px;">Fine Science tools</td>
			<td style="width:119px;">11274-20</td>
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			<td height="42" style="height:42px;width:216px;">Spoon shaped spatula</td>
			<td style="width:112px;">Fine Science tools</td>
			<td style="width:119px;">10090-13</td>
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			<td height="21" style="height:21px;width:216px;">Tisseel Fibin gel kit&#160;</td>
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			<td align="right" style="width:119px;">718971</td>
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			<td height="42" style="height:42px;width:216px;">needle holder</td>
			<td style="width:112px;">Fine Science tools</td>
			<td style="width:119px;">12060-01</td>
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			<td height="21" style="height:21px;width:216px;">vicryl suture 4-0</td>
			<td style="width:112px;">Ethicon</td>
			<td style="width:119px;">J392H</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antisedan</td>
			<td style="width:112px;">Zoetis</td>
			<td style="width:119px;">NADA#141033</td>
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			<td height="21" style="height:21px;width:216px;">Heparin</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">H3393</td>
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			<td height="21" style="height:21px;width:216px;">20ml luerlock&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">23G scalp vein set (butterfly needle)</td>
			<td style="width:112px;">BD</td>
			<td align="right" style="width:119px;">367342</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hemostat</td>
			<td style="width:112px;">Fine Science tools</td>
			<td style="width:119px;">13008-12</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe pump</td>
			<td style="width:112px;">Harvard apparatus</td>
			<td style="width:119px;">PHD 2000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3sec gel glue&#160;</td>
			<td style="width:112px;">Scotch</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rodent dissection board</td>
			<td style="width:112px;">Leica</td>
			<td style="width:119px;">38DI02313</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microfil MV-122</td>
			<td style="width:112px;">flow-tech</td>
			<td style="width:119px;">MV-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">uCT40 scanner</td>
			<td style="width:112px;">Scanco</td>
			<td style="width:119px;">uCT40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCA6%</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">T6399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osteosense 680</td>
			<td style="width:112px;">PerkinElmar</td>
			<td style="width:119px;">NEV10020EX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Angiosense750</td>
			<td style="width:112px;">PerkinElmar</td>
			<td style="width:119px;">NEV10011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxigen 100% medical grade</td>
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			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane (furane)</td>
			<td style="width:112px;">Baxter</td>
			<td align="right" style="width:119px;">1001936040</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IVIS kinetics</td>
			<td style="width:112px;">Xenogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beetle luciferin</td>
			<td style="width:112px;">Promega</td>
			<td style="width:119px;">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

11.3K 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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

将生物探针使用硅烷偶联剂的硅光学生物传感器

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

集成光声眼底镜检查和谱域光学相干断层扫描

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

一种改进的机械性能测试方法来评估骨种植体支抗

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

使用微X射线计算机断层扫描与磷酸制备可视化人类纤维肌组织

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

分离的月毛心室肌细胞的光学成像

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...