这项研究得到了深圳孔雀计划, 中国 (No. 110811003586331), 深圳基础研究项目 (No。JCYJ20150401150223631, No。JCYJ20150401145529020 和 No。JCYJ20160331190714896), 广东省公共研究和能力建设专项计划 (No. 2015A020212030), 国家自然科学基金 (No. 81501893), 中国国家重点基础研究项目 (2013CB945503), 和先进创新计划为优秀青年研究员 (Y5G010)。

动物保育符合广东省实验动物保育和使用指南。所有程序都是在中国科学院深圳高等技术研究院动物研究伦理委员会的监督和批准下进行的. 
 1. 慢 (LV) 生产 <ol> <li> 在自定义多克隆站点的慢表达式向量 (pLenti6/5-eGFP 或 lv-eGFP) 中克隆出一个, 使用 Spe I 和萨尔的巨细胞病毒启动子的下游我限制网站构造 pLenti6/5-PDGFB-eGFP 质粒 (LV- PDGFB ) 25 . </li>
	<li> 慢粒子 (LV- pdgfb ) 从 HEK-293FT 细胞中 co-transfecting 与病毒包装质粒 (pLP1, pLP2, pVSV-G) 使用的标准磷酸钙方法与氯喹 (最终浓度:25 和 #181; M) 25 . </li> <li> 经过48小时的转染、收获和过滤器500毫升的含有病毒的上清液 (0.45 和 #181; m)。随后, 在200和 #181 中, 以 8.9万 x g 为离心, 将病毒颗粒浓缩为2重; 在-80 和 #176, 分和贮存; C. </li>
	<li> 要确定慢的效价, 以连续稀释的慢溶液为24小时孵育 HEK293T 细胞, 并计算慢的效价, 如前面所描述的 25 . </li>
</ol> 2。制备3维印制的 plga/nHAp 支架和 LV 固定化 <ol> <li> 在20毫升 14-二中溶解20克 plga (聚 D, l-丙交酯-co-glycolide) 材料, 形成均匀的溶液。在溶液中加入2克纳米羟基磷灰石 (hydroxyapatide) 粉末 (nHAp);PLGA: nHAp 比应为 10:1 (w/w). </li>
	<li> 用磁力搅拌器将混合溶液强力搅拌 (搅拌速度: 1500 rpm), 室温为 16 h, 形成均匀的糊状。根据预先设计好的模型 26 , 将它制成多孔的 PLGA/nHAp 支架, 使用3D 低温打印机和计算机喷嘴, 将糊层按层、底部沉积到顶部;支架的孔径范围从200到 400 #181; m. </li>
	<li> 真空 freeze-dry 48 h 的支架以尽可能彻底地去除溶剂。将75% 乙醇溶液中的支架浸泡1小时, 进行灭菌和 lyophilize, 以获得中性的无菌支架. 
	注: 真空 freeze-drying 的主要参数包括冷凝温度 (和 #176; C) 和真空度 (Pa)。在真空度为 45 Pa, PLGA/nHAp 支架是真空 freeze-dried 在-78 和 #176; C 为48小时, 以彻底消除溶剂. </li>
	<li> 将10和 #181; L 的 LV 粒子 (4.5 x 10 5 ) 添加到每个 PLGA/nHAp 支架 (4 mm x 4 mm x 2 mm), 并在2和 #176 中孵育 37 h 的支架; C 在湿化孵化器中允许吸附. </li>
	<li> 固定后, 用 PBS 冲洗两次支架以去除未绑定的 LV 颗粒。将 LV 颗粒涂层的支架在液氮中 lyophilize, 再将其储存在-80 和 #176; C, 以备将来使用. </li>
</ol> 3。 体外 从支架和低压转导活性测定的 lv 颗粒释放动力学研究 <ol> <li> 孵育3D 支架 (4 mm x 4 mm x 2 mm) 携带 lv 绿色荧光蛋白 (lv-eGFP, 4.5 x 10 5 LV 粒子) 1 毫升的完全 DMEM 在2.0 毫升低温37和 #176 的小瓶, C, 摇晃5天. </li>
	<li> 每12小时取1毫升样本, 从 12 h 到 120 h post-incubation, 并更换介质。在每个时间点, 增加1毫升的新鲜培养基, 而不是1毫升的孵化培养基. </li>
	<li> 使用收集的介质通过 co-incubating 48 h 来传感器 HEK293T 单元格. 通过流式细胞仪测定荧光蛋白阳性 HEK293T 细胞的数量来测量活性 LV 颗粒的数量 27 . 
	<ol> <li> 在换 HEK293T 单元格之前, 移除培养基, 然后用 PBS 冲洗两次 HEK293T 细胞。添加收集的媒体, 其中含有慢从脚手架释放到井 (1 毫升/井) 和文化与 HEK293T 细胞为48小时, 在37和 #176; C 在湿恒温孵化器. </li>
	</ol> </li> </ol> 4。 体外 骨髓源性 MSC (骨髓) 迁移的评估 <ol> <li> 迁移前, 准备表达番茄荧光蛋白 (骨髓基质细胞 t) 和血小板生长因子 BB 蛋白 (骨髓基质细胞 p) 28 。
	<li> 使用具有8和 #181 孔径的24孔板和聚碳酸酯过滤器对博伊登室执行骨髓迁移试验; m. 
	<ol> <li> 将0.5 毫升的骨髓基质干细胞 (5 x 10 4 ) 转染在上部腔室中, 用 LV 表达番茄荧光蛋白 (骨髓基质细胞 t)。将0.5 毫升骨髓基质干细胞 (2 x 10 5 ) 转染于 LV, 在下部腔内表达血小板生长因子-BB 蛋白 (骨髓基质细胞 P). </li>
	</ol> </li> <li> 在本实验中包含六组, 并添加500和 #181; L 到每个井的下部隔间. 
	a. 空白控制, 无血清培养基只有 B. FBS 控制, 培养基辅以 10% FBS C. 血小板生长因子-bb 控制, 4 ng/毫升-bb 在无血清培养基中 D. 血小板释放-bb (4 ng/毫升) 和抗血小板-bb (4 ng/毫升) 多克隆抗体无血清培养基 E. 骨髓组, 2 x 10 5 无血清培养基中的骨髓基质细胞, 2 x 10 5 骨髓基质细胞-p 在血清自由培养基中。
	<li> 在加湿的恒温箱中孵育48小时, 在 5% CO 2 和37和 #176; C。然后, 移除下腔侧的迁移细胞, 并通过流式细胞术对骨髓基质 t 的数量进行采集. 
	<ol> <li> 使用单元刮板移除下部腔室侧的迁移细胞, 并重2毫升 PBS 的迁移细胞。通过流式细胞术对移植的骨髓基质干细胞数量进行量化 29 . </li>
	</ol> </li> </ol> 5。小鼠颅骨临界骨缺损模型及支架移植的建立 注意: 从广东省医学实验动物中心购买了 balb/c (7 周大) 的雄性鼠. 
<ol> <li> 为颅骨骨缺损试验, 总共招收42只雄性小鼠。随机将小鼠分成三组, 每组中有十四个接受以下治疗: A 组, 无植入物;B 组, PH 支架植入 (PLGA/nHAp);和 C 组, 植入的 PHp 支架 (PLGA/nHAp/LV- pdgfb ). </li>
	<li> 麻醉的动物与戊巴比妥钠腹腔管理 (50 毫克/千克)。在进行第一次切口前, 用脱毛膏去除毛皮, 用75% 乙醇溶液清洁头部皮肤。用立体定向仪固定每只老鼠的头部, 在手术中保持静止. </li>
	<li> 用手术刀做一个初始切口, 然后放大它的机智h 剪刀创建一个1厘米的线性皮肤切口。用无菌棉签从颅骨骨中刮出骨膜, 以露出颅骨的骨表面. </li>
	<li> 使用环毛刺在颅的左侧创建4毫米直径的 critical-size 缺陷 30 。将支架移植到骨缺损部位. 
	注意: 临界尺寸缺陷 (csd) 最初定义为和 #34; 最小尺寸的骨内伤口在特定的骨骼和动物的物种, 不会自发愈合在动物的一生中. #34; 在这个实验中, 4 毫米直径的缺陷是一个关键的大小骨缺损, 根据以前的研究 31 , 32 . </li>
	<li> 用眼科钳将骨膜轻轻移回手术部位。缝合切割皮肤三针每厘米. </li>
	<li> 执行后护理。管理镇痛药丁丙诺啡 (0.1 毫克/千克), 以减轻疼痛。用加热垫保持体温, 直到动物苏醒。随后, 用食物和水喂养动物, 每天记录他们的活动, 直到测试结束。注: 根据当地动物保育委员会的指导方针, 可以在手术前进行镇痛. 和 #160; </li> </ol> 6。 在体内 MPM 骨缺损内血管生成的影像学研究 注意: FITC-共轭 250 kD 葡聚糖 (10 毫克/毫升) 在生理盐水中静脉注射, 以获得高信噪比 (信噪比) 新血管的图像根据先前的研究 33 。#160;P 租约注意, 这是一个生存程序. #160; <ol> <li> 为双光子激发荧光 (TPEF) 和第二次谐波信号 (倍) 成像组装多显微镜 (MPM) 系统. </li>
	<li> 麻醉用腹腔注射戊巴比妥钠 (50 毫克/千克) 将动物固定在加热板上, 以保持其体温在37和 #176; C 贯穿整个实验。在100% 氧气中使用3.0% 的异氟醚气体, 在成像过程中保持满意的麻醉和镇痛效果。腹腔注射生理盐水 (200 #181; 动物), 以防止在成像前脱水. </li>
	<li> 将飞秒钛蓝宝石激光器调至 860 nm。创建一个 512 x 512 和 #181; m 采样区域通过扫描一对振镜和使用 NA1.0 浸水物镜 focusthe 励磁光束到样品和收集散射 TPEF/倍信号. 
	注: 成像是在 home-build 多显微成像系统 (MPM) 上进行的。在成像过程中, 一颗飞秒钛蓝宝石激光器被调谐到 860 nm 作为最佳激励波长。激光光束是光栅扫描, 通过一个样本平面, 使用了一对电流计镜。经过 685 nm 的分色镜, 光束被聚焦在标本上的 20X NA1.0 目标。TPEF 和倍信号是由同一目标收集的。上述信号从激发激光器中分离出来, 由 680 nm 的短路过滤器净化。TPEF 和倍信号由纤维束收集并进行光谱仪。光谱仪上的探测器是光电倍增管 (并联) 的线性阵列。光谱仪和线性阵列并联的组合提供了记录信号的能力, 在16连续的波段, 从 400 nm 到 600 nm, 以 12.5 nm 的间隔。因此, 同时检测 TPEF 和倍信号, 并在谱域中分离。此外, 对于3D 成像, 用轴向马达来控制成像深度。每个图像的面积设置为 512 x 512 和 #181; m, 深度间隔设置为2和 #181; m (控制组, 5 和 #181; m)。值得注意的是, 每个缺陷上的5站点都被成像以收集有统计学意义的数据, 而所选的成像站点则沿缺陷均匀随机分布. </li>
	<li> 扫描每个缺陷上的5站点, 以收集具有统计学意义的数据来测量容器的形成. 
	<ol> <li> 将扫描站点均匀地使用, 并沿缺陷随机分布。对于对照组, 有视野 (视) 覆盖原骨区和边缘的缺陷, 但对于 PH 和 PHp 组, 有视在植入支架上. </li>
		<li> 做一个1厘米的线性皮肤切口使用手术刀和缝合开放皮肤的固定, 以揭示移植网站。使用注射器在浸渍透镜和移植之间放置一滴水水来形成水玻璃. </li>
	</ol> </li> <li> 每年十二年代在2小时成像期间获取图像堆栈。使用自定义的 MATLAB 程序显示和分析体积数据, 并用 ImageJ 软件 34 , 35 来量化血管区域. </li>
</ol> 7。qPCR 的基因表达分析及与血管生成相关基因的研究 注意: pcr 是按照通常的步骤执行的:95 和 #176; c 为三十年代, 40 周期95和 #176; c 为 5 s, 和60和 #176; c 为三十年代. pcr 后的熔化曲线证实了单扩增的特异性, 以及与 #946 相关的兴趣基因的褶皱变化;对每个样品的反应进行了三次测试. 
<ol> <li> 从实验小鼠处采集植入的支架和组织, 在2、4、8周后进行 36 ; 对照组标本 (n = 4) 同时服用, 应从相邻的骨组织中提取。
	注意: 在每一个时间点 (2, 4, 和8周), 安乐死的小鼠与 CO 2 吸入。解剖的颅, 并随后, 删除和收获的植入支架从颅. </li>
	<li> 根据制造商和 #39 的协议, 使用商用试剂从每个样品中提取总 RNA。使用反向转录试剂盒, 将 RNA 转录到制造商和 #39 的协议之后. </li>
	<li> 使用 SYBR 绿色检测系统执行定量 real-time PCR; 该底漆列在 表 1 中. </li>
</ol> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"> <tbody> <tr> <td colspan="8"> 入门 (5 和 #39; #8211; 3 和 #39;) </td> </tr> <tr> <td colspan="2" rowspan="1"> 基因 </td>
			<td colspan="2" rowspan="1"> 转发 </td>
			<td colspan="4" rowspan="1"> 反向 </td> </tr>
		<tr> <td> pdgfb </td> <td colspan="3"> CATCCGCTCCTTTGATGATCTT </td> <td colspan="4"> GTGCTCGGGTCATGTTCAAGT </td> </tr>
		<tr> <td> vWF </td> <td colspan="3"> CTCTTTGGGGACGACTTCATC </td> <td colspan="4"> TCCCGAGAATGGAGAAGGAAC </td> </tr>
		<tr> <td> VEGFR2 </td> <td colspan="3"> GAAATGACACTGGAGCCTACA AG </td> <td colspan="4"> TCCATGCTGGTCACTAACAGAA G </td> </tr>
		<tr> <td> 和 #946;-肌动蛋白 </td> <td colspan="3"> GTATCCATGAAATAAGTGGTTAC AGG </td> <td colspan="4"> GCAGTACATAATTTACACAGAAG 审计 </td> </tr> </tbody>
</table> 表 1: Pimer 集. 
 8. 骨再生的 MicroCT 分析 <ol> <li> 安乐在术后8周吸入 CO 2 的小鼠。在通风良好的环境中, 将每只老鼠轻轻地放入一个干净的老鼠笼中, 泵 CO 2 气体进入笼子, 在安乐死前麻醉小鼠. </li>
	<li> 解剖颅骨以 microCT 骨缺损区的影像。用户在实验中的以下扫描参数: 9 和 #181; m 分辨率, Al 0.5 mm 滤波器, 50 伏电压, 142 和 #181; 电流. </li>
	<li> 侦察使用公司提供的商业软件对所有成像数据进行结构设计。使用 CTAn 软件的校准功能校准扫描. 
	注: 在每次扫描中, 在鼠标下放置一个 Hounsfield 单元 (HU). </li>
</ol>

<ol>
	<li>Hu, 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=GPNMB+enhances+bone+regeneration+by+promoting+angiogenesis+and+osteogenesis:+potential+role+for+tissue+engineering+bone.">GPNMB enhances bone regeneration by promoting angiogenesis and osteogenesis: potential role for tissue engineering bone.</a> J Cell Biochem. 114 (12), 2729-2737 (2013).</li><li>Schroeder, J. E., Mosheiff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+approaches+for+bone+repair:+concepts+and+evidence.">Tissue engineering approaches for bone repair: concepts and evidence.</a> Injury. 42 (6), 609-613 (2011).</li><li>Chiarello, E., 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=allograft+and+bone+substitutes+in+reconstructive+orthopedic+surgery.">allograft and bone substitutes in reconstructive orthopedic surgery.</a> Aging Clin Exp Res. 25, S101-S103 (2013).</li><li>Elangovan, 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=The+enhancement+of+bone+regeneration+by+gene+activated+matrix+encoding+for+platelet+derived+growth+factor.">The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor.</a> Biomaterials. 35 (2), 737-747 (2014).</li><li>Bouyer, 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=Surface+delivery+of+tunable+doses+of+BMP-2+from+an+adaptable+polymeric+scaffold+induces+volumetric+bone+regeneration.">Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration.</a> Biomaterials. 104, 168-181 (2016).</li><li>Rezwan, 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=Biodegradable+and+bioactive+porous+polymer/inorganic+composite+scaffolds+for+bone+tissue+engineering.">Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering.</a> Biomaterials. 27 (18), 3413-3431 (2006).</li><li>Burg, K. J., Porter, S., Kellam, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+developments+for+bone+tissue+engineering.">Biomaterial developments for bone tissue engineering.</a> Biomaterials. 21 (23), 2347-2359 (2000).</li><li>Xiao, Y., 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=Modifications+of+collagen-based+biomaterials+with+immobilized+growth+factors+or+peptides.">Modifications of collagen-based biomaterials with immobilized growth factors or peptides.</a> Methods. 84, 44-52 (2015).</li><li>Chen, G., Lv, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+and+Application+of+Electrospun+Nanofiber+Scaffold-based+Growth+Factor+in+Bone+Tissue+Engineering.">Immobilization and Application of Electrospun Nanofiber Scaffold-based Growth Factor in Bone Tissue Engineering.</a> Curr Pharm Des. 21 (15), 1967-1978 (2015).</li><li>Kofron, M. D., Li, X., Laurencin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-+and+gene-based+tissue+engineering+in+bone+repair.">Protein- and gene-based tissue engineering in bone repair.</a> Curr Opin Biotechnol. 15 (5), 399-405 (2004).</li><li>Chen, F. 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=New+insights+into+and+novel+applications+of+release+technology+for+periodontal+reconstructive+therapies.">New insights into and novel applications of release technology for periodontal reconstructive therapies.</a> J Control Release. 149 (2), 92-110 (2011).</li><li>Winn, S. R., 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=Gene+therapy+approaches+for+modulating+bone+regeneration.">Gene therapy approaches for modulating bone regeneration.</a> Adv Drug Deliv Rev. 42 (1-2), 121-138 (2000).</li><li>Chang, P. 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=Adenovirus+Encoding+Human+Platelet-Derived+Growth+Factor-B+Delivered+to+Alveolar+Bone+Defects+Exhibits+Safety+and+Biodistribution+Profiles+Favorable+for+Clinical+Use.">Adenovirus Encoding Human Platelet-Derived Growth Factor-B Delivered to Alveolar Bone Defects Exhibits Safety and Biodistribution Profiles Favorable for Clinical Use.</a> Hum Gene Ther. 20 (5), 486-496 (2009).</li><li>Phipps, M. C., Xu, Y. Y., Bellis, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+Platelet-Derived+Growth+Factor+as+a+Chemotactic+Factor+for+Mesenchymal+Stem+Cells+by+Bone-Mimetic+Electrospun+Scaffolds.">Delivery of Platelet-Derived Growth Factor as a Chemotactic Factor for Mesenchymal Stem Cells by Bone-Mimetic Electrospun Scaffolds.</a> Plos One. 7 (7), (2012).</li><li>Gehmert, 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=Angiogenesis:+The+role+of+PDGF-BB+on+Adiopse-tissue+derived+Stem+Cells+(ASCs).">Angiogenesis: The role of PDGF-BB on Adiopse-tissue derived Stem Cells (ASCs).</a> Clin Hemorheol and Microcirc. 48 (1-3), 5-13 (2011).</li><li>Chang, P. 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=PDGF-B+gene+therapy+accelerates+bone+engineering+and+oral+implant+osseointegration.">PDGF-B gene therapy accelerates bone engineering and oral implant osseointegration.</a> Gene Ther. 17 (1), 95-104 (2010).</li><li>Javed, F., 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=Significance+of+the+platelet-derived+growth+factor+in+periodontal+tissue+regeneration.">Significance of the platelet-derived growth factor in periodontal tissue regeneration.</a> Arch Oral Biol. 56 (12), 1476-1484 (2011).</li><li>Murali, R., 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=Biomimetic+hybrid+porous+scaffolds+immobilized+with+platelet+derived+growth+factor-BB+promote+cellularization+and+vascularization+in+tissue+engineering.">Biomimetic hybrid porous scaffolds immobilized with platelet derived growth factor-BB promote cellularization and vascularization in tissue engineering.</a> J Biomed Mater Res A. 104 (2), 388-396 (2016).</li><li>Andrae, J., Gallini, R., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+platelet-derived+growth+factors+in+physiology+and+medicine.">Role of platelet-derived growth factors in physiology and medicine.</a> Genes Dev. 22 (10), 1276-1312 (2008).</li><li>Wosnitza, 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=Plasticity+of+human+adipose+stem+cells+to+perform+adipogenic+and+endothelial+differentiation.">Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation.</a> Differentiation. 75 (1), 12-23 (2007).</li><li>Hankenson, K. 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=Angiogenesis+in+bone+regeneration.">Angiogenesis in bone regeneration.</a> Injury. 42 (6), 556-561 (2011).</li><li>Schmidt, 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=Rapid+three-dimensional+quantification+of+VEGF-induced+scaffold+neovascularisation+by+microcomputed+tomography.">Rapid three-dimensional quantification of VEGF-induced scaffold neovascularisation by microcomputed tomography.</a> Biomaterials. 30 (30), 5959-5968 (2009).</li><li>Perng, C. 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=In+Vivo+Angiogenesis+Effect+of+Porous+Collagen+Scaffold+with+Hyaluronic+Acid+Oligosaccharides.">In Vivo Angiogenesis Effect of Porous Collagen Scaffold with Hyaluronic Acid Oligosaccharides.</a> J Surg Res. 168 (1), 9-15 (2011).</li><li>Sun, Y., 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=Imaging+tissue+engineering+scaffolds+using+multiphoton+microscopy.">Imaging tissue engineering scaffolds using multiphoton microscopy.</a> Microsc Res Tech. 71 (2), 140-145 (2008).</li><li>Fan, 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=Noninvasive+monitoring+of+placenta-specific+transgene+expression+by+bioluminescence+imaging.">Noninvasive monitoring of placenta-specific transgene expression by bioluminescence imaging.</a> PLoS One. 6 (1), e16348 (2011).</li><li>Yeo, M. G., Kim, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Characterization+of+3D+Composite+Scaffolds+Based+on+Rapid-Prototyped+PCL/&#946;-TCP+Struts+and+Electrospun+PCL+Coated+with+Collagen+and+HA+for+Bone+Regeneration.">Preparation and Characterization of 3D Composite Scaffolds Based on Rapid-Prototyped PCL/&#946;-TCP Struts and Electrospun PCL Coated with Collagen and HA for Bone Regeneration.</a> Chem Mater. 24 (5), 903-913 (2012).</li><li>Mao, Y., 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=Lentiviral+Vectors+Mediate+Long-Term+and+High+Efficiency+Transgene+Expression+in+HEK+293T+cells.">Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells.</a> Int J Med Sci. 12 (5), 407-415 (2015).</li><li>Li, J., 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=Investigation+of+angiogenesis+in+bioactive+3-dimensional+poly+(D,L-lactide-co-glycolide)/nano-hydroxyapatite+scaffolds+by+in+vivo+multiphoton+microscopy+in+murine+calvarial+critical+bone+defect.">Investigation of angiogenesis in bioactive 3-dimensional poly (D,L-lactide-co-glycolide)/nano-hydroxyapatite scaffolds by in vivo multiphoton microscopy in murine calvarial critical bone defect.</a> Acta Biomater. 42, 389-399 (2016).</li><li>Abbasi, 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=Lentiviral+vector-mediated+transduction+of+goat+undifferentiated+spermatogonia.">Lentiviral vector-mediated transduction of goat undifferentiated spermatogonia.</a> Anim Reprod Sci. 163, 10-17 (2015).</li><li>Pigossi, S. 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=Bacterial+cellulose-hydroxyapatite+composites+with+osteogenic+growth+peptide+(OGP)+or+pentapeptide+OGP+on+bone+regeneration+in+critical-size+calvarial+defect+model.">Bacterial cellulose-hydroxyapatite composites with osteogenic growth peptide (OGP) or pentapeptide OGP on bone regeneration in critical-size calvarial defect model.</a> J Biomed Mater Res A. , (2015).</li><li>Bos, G. 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=The+effect+of+histocompatibility+matching+on+canine+frozen+bone+allografts.">The effect of histocompatibility matching on canine frozen bone allografts.</a> J Bone Joint Surg Am. 65 (1), 89-96 (1983).</li><li>Hollinger, J. O., Kleinschmidt, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+critical+size+defect+as+an+experimental+model+to+test+bone+repair+materials.">The critical size defect as an experimental model to test bone repair materials.</a> J Craniofac Surg. 1 (1), 60-68 (1990).</li><li>Hollanders, 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=Bevacizumab+Revisited:+Its+Use+in+Different+Mouse+Models+of+Ocular+Pathologies.">Bevacizumab Revisited: Its Use in Different Mouse Models of Ocular Pathologies.</a> Curr Eye Res. 40 (6), 611-621 (2015).</li><li>Gao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QSIM:+quantitative+structured+illumination+microscopy+image+processing+in+ImageJ.">QSIM: quantitative structured illumination microscopy image processing in ImageJ.</a> Biomed Eng Online. 14, 4 (2015).</li><li>Kobat, 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=Deep+tissue+multiphoton+microscopy+using+longer+wavelength+excitation.">Deep tissue multiphoton microscopy using longer wavelength excitation.</a> Opt Express. 17 (16), 13354-13364 (2009).</li><li>Lohmann, 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+regeneration+induced+by+a+3D+architectured+hydrogel+in+a+rat+critical-size+calvarial+defect.">Bone regeneration induced by a 3D architectured hydrogel in a rat critical-size calvarial defect.</a> Biomaterials. 113, 158-169 (2016).</li><li>Lv, J., 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=Enhanced+angiogenesis+and+osteogenesis+in+critical+bone+defects+by+the+controlled+release+of+BMP-2+and+VEGF:+implantation+of+electron+beam+melting-fabricated+porous+Ti6Al4V+scaffolds+incorporating+growth+factor-doped+fibrin+glue.">Enhanced angiogenesis and osteogenesis in critical bone defects by the controlled release of BMP-2 and VEGF: implantation of electron beam melting-fabricated porous Ti6Al4V scaffolds incorporating growth factor-doped fibrin glue.</a> Biomed Mater. 10 (3), (2015).</li><li>Guldberg, R. E., 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=3D+imaging+of+tissue+integration+with+porous+biomaterials.">3D imaging of tissue integration with porous biomaterials.</a> Biomaterials. 29 (28), 3757-3761 (2008).</li><li>Boehler, R. 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=A+PLG/HAp+composite+scaffold+for+lentivirus+delivery.">A PLG/HAp composite scaffold for lentivirus delivery.</a> Biomaterials. 34 (21), 5431-5438 (2013).</li><li>Heo, S. J., 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=Fabrication+and+characterization+of+novel+nano-+and+micro-HA/PCL+composite+scaffolds+using+a+modified+rapid+prototyping+process.">Fabrication and characterization of novel nano- and micro-HA/PCL composite scaffolds using a modified rapid prototyping process.</a> J Biomed Mater Res A. 89 (1), 108-116 (2009).</li></ol>

作者声明他们没有竞争的金融利益。

骨是一种高度带血管的组织, 具有独特的能力, 可以在单个1的整个生命周期中持续地进行修复和重塑。血管化水平对成骨和缺损修复具有重要意义。低血管化限制了组织工程骨的广泛临床应用。根据仿生学的理论构建高度血管化的组织工程骨, 已成为修复大段骨缺损的工具。各种支架已成功地用于修复比较小的骨缺损。然而, 对于严重的骨缺损, 应用支架修复仍然面临的障碍, 主要是由于缺乏足够的血液供应缺陷修复。因此, 在修复大骨缺损的过程中, 改善血液供应是组织工程中的主要问题之一。
人们使用生物活性材料涂层支架, 以帮助建立必要的形态发生的领域, 可以刺激, 招募, 并促进细胞的趋化, 分化和增殖所需的新生和骨形成37. 新生在组织工程支架中的作用是细胞接收氧气和养分, 去除废品, 最终实现支架功能的必要条件。本研究的目的是探讨血管生成与体内多显微镜在一个基因修饰, 3 d 印制 PLGA/nHAp 支架颅骨关键骨缺损修复和评价的影响, 骨再生利用microCT
尽管在骨组织工程方面取得了巨大的成就, 但在骨再生过程中, 支架内的血管生成的活体可视化和量化仍然是一个挑战。大多数显微成像系统无法提供关于血管生成的体积信息。传统的成像方法, 如 microCT, MRI, 组织学分析, 是破坏性的, 复杂的, 费力的, 具有低的空间分辨率和长的图像采集时间38。然而, MPM 成像是一种新的生物成像模式, 可以捕获血管信号在体内以高分辨率和微创的方式。它能够检测出比10µm 更薄的新容器 (图 4B)。除了血管图像, 我们还观察到了大量的新的胶原沉积 (图4B 中的绿色颜色和补充材料中的 GIF 图), 如倍信号表明, 从另一个角度表明骨组织的形成。从理论上讲, 可以在不使用造影剂的情况下获得血管图像。然而, 在本研究中, 我们使用了造影剂, 以获得更好的图像和删除局部皮肤, 以确保正确的扫描深度。这将是不必要的, 当更强大的成像系统, 在未来的真正非侵入成像。
HAp 是一种用于骨组织再生的生物医学材料, 由于其良好的传导和成特性而被广泛使用。HAp 具有良好的结合 DNA 载体和保持稳定载体的能力, 包括病毒载体39。与微型 hap 相比, 纳米羟基磷灰石粒子具有较高的比表面积, 因此表现出较好的细胞行为和力学性能40。由于这些原因, nHAp 粒子是脚手架的组成部分之一。图1所示的 PLGA/nHAp 支架的表面和横断面 SEM 图像表明, 所制备的支架具有 well-interconnected 孔结构和均匀分布的孔隙大小。将病毒固定在生物材料载体上可以影响病毒的活性。因此, 我们通过调查其转染活性, 测试了从支架中释放的 LV 微粒的生物活性。从支架和他们的生物活性的 lv 微粒的发行外形被评估了在体外, 展示五天 (图 2) 的被控制的交付活性 lv 微粒。此外, 我们的体外研究重点研究了血小板生长因子 BB 的潜能, 以刺激原发性骨髓间充质干细胞的迁移。这些结果表明, LV 粒子表达了生物活性的血小板生长因子 BB 和有效刺激骨髓迁移。
为了确定血管生成和骨再生能力在体内, PHp 支架植入小鼠颅骨关键骨缺损。血管形成与血管生成标记, RT qPCR 分析, 血管密度观察倍/TPEF 成像。两组实验均表明, 随着转基因支架的处理, 血管生成和血管形成明显改善。这些结果表明, 血小板生长因子-BB 不仅直接刺激血管生成, 而且还诱导其他血管生成相关的基因。同时, microCT 的测量表明, 与未修改的支架相比, 血小板生长因子b包含的支架能显著促进骨形成, 这与支架中的新生很好的关联。这些结果表明, 基因修饰, 生物活性支架显著改善血管生成和组织再生。
研究结果表明, 慢介导的支架基因修饰有助于小鼠颅骨临界骨缺损模型的大骨缺损修复, 可能通过改善血管生成。此外,在体内MPM 成像提供了一个独特的工具来理解骨支架血管生成和成骨, 并能够进一步阐明是否有一定的支架有利于新生血管。活体多显微镜成像为了解组织和支架血管生成的生理学和病理生理学提供了一种模式。但是, 使用此技术时有几个限制。首先, 外科手术的准备需要特殊的专业知识。不熟练操作引起的炎症会显著降低 MPM 成像质量。其次, 由于 MPM 成像速度的限制, 需要一个定制设计的鼠标头, 以减少由心跳和呼吸引起的工件。第三, 为了长期成像, 动物必须腹腔或静脉注射生理盐水, 以防止脱水。

骨骼是一种高度带血管的组织, 在单个1的生存期内继续进行重塑。由于外伤、骨不连、肿瘤切除或颅面畸形而导致的大骨缺损的快速有效的骨再生是一个复杂的生理过程。传统的治疗方法用于骨缺损修复包括自体移植和同种异体植入, 但其使用涉及几个问题和限制, 如有限的可用性, 严重的供者部位的发病率, 高的感染风险, 和主机免疫排斥2,3。然而, 人工骨移植提供了一种有效的替代方法来缓解这些局限性。它们可以由可生物降解的材料制成, 很容易制造出合适的孔径, 并可进行基因改造4,5。
目前, 在组织工程骨的开发中, 使用了各种组织工程学支架6,7。为了更有效地诱导骨修复和再生, 与生长因子结合的工程生物材料已经出现并取得了良好的效果8,9。不幸的是, 短暂的半衰期, easy-to-lose 活性, 和 supraphysiological 剂量的生长因子的治疗效果限制他们的临床应用10。为了克服这些问题, 将生长因子基因的传递, 而不是生长因子作为一种有效的方法来维持生物活性的治疗骨缺损和疾病11,12。病毒载体是有前途的传递工具, 组织再生由于其高表达效率13。
在生长因子中, 血小板源性生长因子在本研究中被选择, 因为它不仅是间充质和成骨细胞的丝裂原和趋化蛋白, 而且还是一种刺激血管生成的14,15.前期临床研究表明, 血小板生长因子-BB 能安全有效地促进牙周骨缺损的修复,16,17。最近的研究表明, 血小板生长因子-BB 刺激血管内皮细胞迁移和增殖在体内18,19。此外, 血小板生长因子 BB 还能使骨髓间充质干细胞 (mscs) 分化为内皮细胞20, 这进一步凸显了 mscs 在新生血管中的潜在作用。因此, 在骨组织工程支架中, 诱导血管的从头开始形成是修复组织的重要步骤。
骨缺损愈合是一种动态组织形态发生过程, 需要协调成骨和血管生成在修复位置21。血管植入组织工程支架是一个必要的先决条件, 为细胞提供养分和氧气的生长和生存和消除代谢废物。常用的成像方法, 包括 X 射线显微计算机断层扫描 (microCT), 磁共振成像 (MRI), 扫描电子显微镜 (SEM), 光学相干断层扫描 (OCT), 和共焦激光扫描显微镜, 而不是使用组织学检查获取血管生成信息22,23。然而, 这些方法在骨组织工程3D 支架的可视化和测量新生中面临各种障碍。多显微镜 (MPM) 是一种比较新颖的生物成像技术, 具有明显的优势, 同时可视化细胞, 细胞外基质, 和周围的血管网络在体内.它具有一个固有的三维成像能力, 深层组织穿透和导致低光。因此, 在过去的十年中, MPM 在生物医学研究24中得到了很大的关注, 包括神经科学、免疫学和干细胞动力学。然而, 它几乎没有用于骨科研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly(D,L-lactide-co-glycolide) (PLGA)</td>
			<td>Sigma</td>
			<td>P1941</td>
			<td>L/G ratio 75:25, MW 66000-107000</td>
		</tr>
		<tr>
			<td>Hydroxyapatite nanoparticles</td>
			<td>Sigma</td>
			<td>702153</td>
			<td>Average diameter &#60; 200nm</td>
		</tr>
		<tr>
			<td>Chloroquine diphosphate salt</td>
			<td>Sigma</td>
			<td>C6628</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated 250-kD dextran</td>
			<td>Sigma</td>
			<td>FD250S</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-dioxane</td>
			<td>lingfeng,Shanghai</td>
			<td></td>
			<td>0.45 micron</td>
		</tr>
		<tr>
			<td>Stericup filters</td>
			<td>Merck Millipore Corporation</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB Cdna</td>
			<td>Sino Biological, Inc</td>
			<td>MZ50801-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PDGF-BB mouse polyclonal antibody</td>
			<td> BioVision, Inc</td>
			<td>5489-30T</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB recombinant protein</td>
			<td></td>
			<td>4489-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium-phosphate transfection solution</td>
			<td>Promega Corporation</td>
			<td>E1200</td>
			<td></td>
		</tr>
		<tr>
			<td>L-DMEM</td>
			<td>Hyclone</td>
			<td>SH30021.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Hyclone</td>
			<td>SH30028.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, Liquid</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell </td>
			<td>Corning</td>
			<td>3422</td>
			<td></td>
		</tr>
		<tr>
			<td>Male BALB/c mice</td>
			<td>Guangdong Medical Laboratory Animal Center </td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pentobarbital </td>
			<td>Merck</td>
			<td>1063180500</td>
			<td></td>
		</tr>
		<tr>
			<td>multiphoton microscopy</td>
			<td></td>
			<td></td>
			<td>A homemade in Shenzhen Institutes of Advanced Technology to detect two-photon excited fluorescence (TPEF) and second harmonic generation signal (SHG).</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Keyuan, Shandong</td>
			<td>401750169</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript RT Master Mix (Perfect Real Time)</td>
			<td>Takara</td>
			<td>RR420B</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Premix Ex Taq (Tli RNaseH Plus)</td>
			<td>Takara</td>
			<td>RR036B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin</td>
			<td>Beyotime</td>
			<td>C0105</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Leica </td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge OPtima L-100XP</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-temperature printer </td>
			<td>Tsinghua university</td>
			<td></td>
			<td>A homemade in Tsinghua university</td>
		</tr>
		<tr>
			<td>LightCycler 480 instrument </td>
			<td>Roche</td>
			<td>5815916001</td>
			<td></td>
		</tr>
		<tr>
			<td>microCT</td>
			<td>Bruker</td>
			<td>1176</td>
			<td></td>
		</tr>
		<tr>
			<td>commercial software</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing angiogenesis by multiphoton microscopy in vivo in genetically modified 3d-plga/nhap scaffold for calvarial critical bone defect repair

重度骨缺损的重建仍然是一个严重的临床问题, 因为在修复过程中组织工程支架内血管生成不良, 导致缺乏足够的血液供应, 造成新组织坏死。快速血管化是新的组织生存和与现有宿主组织融合的重要先决条件。在脚手架上的从头到脚生成是使骨骼再生更有效的最重要步骤之一, 允许修复组织成长为支架。为了解决这个问题, 生物材料支架的基因修饰用于加速血管生成和成骨。然而, 在 real-time 和三维 (3D) 支架或新骨组织中, 可视化和追踪体内血管形成仍然是骨组织工程的障碍。多显微镜 (MPM) 是一种新的生物成像模式, 可以获得高分辨率和微创的方式从生物结构的体积数据。本研究的目的是在一个基因修饰的 3 d-PLGA/nHAp 支架中多显微镜下观察血管生成, 用于颅骨临界骨缺损修复. PLGA/nHAp 支架功能化, 以持续交付一个生长因子血小板增殖素 b基因携带慢载体 (LV-pdgfb), 以促进血管生成和促进骨骼再生。在支架植入的颅骨临界骨缺损小鼠模型中, PHp 支架内的血管区 (BVAs) 明显高于 PH 支架。此外, 血小板生长因子-b和血管生成相关基因的表达, vWF和VEGFR2也相应增加。MicroCT 分析表明, 与其他组相比, PHp 组的新骨形成明显改善。据我们所知, 这是第一次用多显微镜进行骨组织工程, 以研究3D 生物降解支架内的血管生成,在体内和 real-time。

用3D 打印机制作了0.6 毫米高和直径4毫米的圆柱形多孔 PLGA/nHAp 支架。通过扫描电镜和 microCT 对支架的形态进行了分析。图 1A显示了植入支架的照片。MicroCT 扫描显示, 超过85% 的毛孔有大小不等, 从200到400µm (图 1B)。SEM 成像表明, 脚手架的表面有一个粗略的形貌, 与微 (直径约 5-10 µm), 在脚手架内互联 (图 1 d-F)。从 PLGA/nHAp 支架中释放出的 eGFP 颗粒 (4.5 x 105) 的总含量中, 大约40% 是在 24 h 内开始的初始爆裂效应。释放的 LV 病毒的生物活性到达了山顶在 24 h 然后连续地被减少, 接近到0在 120 h (图 2)。这些数据表明, 大多数支架固定化的 LV 颗粒的生物活性可维持 96 h。采用细胞分析方法观察了骨髓基质干细胞的迁移能力, 探讨了低压携带血小板生长因子b cDNA 是否能表达并导致骨髓趋化, 如图 3所示。阳性对照组 (C) 和血小板生长因子-BB 表达群 (F) 的骨髓迁移水平均明显高于其他4组。此外, F 组骨髓基质细胞的迁移明显高于 C 组。其他4组间的骨髓迁移没有显著差异。
骨缺损的血管生成从第二到第八周 post-implantation MPM 扫描评估。图 4A显示了 MPM 扫描下鼠标的示意图。在 PH 和 PHp 组中发现了明显的血管生成, 但在对照组中血管几乎没有观察到, 仅在缺损区形成一层纤维膜 (图 4B)。GIF 图显示每个组的血管 tomographs (补充数据)。随着时间的推移, 新血管的面积逐渐增加, 其 PH 值和 PHp 也有相似的增长模式。在第八周, 血管形态与早期观察到的相似;唯一的变化是越来越多的小型船只成长为脚手架 (图 4B)。在所有时间点 (图 4C) 中, PHp 组的 BVA 明显高于其他两个组。此外, 还有许多胶原沉积点, 如倍信号 (图和 GIF 图中的绿色颜色) 在 PH 值和 PHp 组中显示, 但在对照组中不存在 (图 4B)。
为了阐明血小板生长因子-BB 表达与血管生成的关系, 我们将血小板源性 b、 vWF和VEGFR2的基因转录表达水平与 RT qPCR 方法在2、4和8周 post-implantation (图 5)。如预期的那样, 血小板生长因子-b表达式在 PHp 组中的所有时间点都显著增加, 与控件和 PH 值组相比, 在5到13倍的增量 (图 5A)。对照组与 PH 值之间无显著性差异, 而血小板生长因子 BB 的表达水平从2周到8周几乎保持不变, 如图 5A所示。所有三组中vWF和VEGFR2的 mRNA 表达式级别逐渐增加, 并且在所有三时间点 (图 5B和5C) 中都是 PHp 组中最高的。对于 PH 值组, 尽管有明显的血管生成 (图 4B), 但与对照组相比没有统计学意义。
为了进一步确认血小板生长因子b-含支架促进骨骼再生在体内使用严重大小的颅骨缺陷的男性 balb/c/c 小鼠, microCT 扫描提供的信息评估骨再生8周post-implantation如图 6所示, PHp 组中的缺陷在支架中填充了大量新的骨组织长, 而在控制和 PH 值组中的新骨量则少得多。
<img alt="Figure 1" src="/files/ftp_upload/55381/55381fig1.jpg"> 
图 1: 3D PLGA/nHAp 支架的结构特征.(a) 3D 印花脚手架的照片。(B) 脚手架整体孔隙度的 microCT 图像。(C) 在60X、2,000X (D)、5,000X (E) 和10,000X 放大 (F) 上观察到的支架表面的形貌。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55381/55381fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" src="/files/ftp_upload/55381/55381fig2.jpg"> 
图 2: 制备慢和评价 LV-GFP 粒子的生物活性.(A) 慢向量结构的示意图表示形式。在 48 h 转染后, HEK-293FT 细胞在光 (B) 和荧光场 (C) 条件下观察。(D)体外评估从 PLGA/nHAp 支架中累计释放的 LV-GFP 粒子的生物活性。所有数据均以平均± SEM (n = 4) 显示。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55381/55381fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" src="/files/ftp_upload/55381/55381fig3.jpg"> 
图3。血小板生长因子-BB 生物活性作为骨髓迁移的一个有力的趋因素反应。骨髓基质细胞 t (5 x 104) 被播种在 transwell 室的顶部, 各种条件介质放置在腔室的底部。( A) 空白控件, 仅限无血清培养基;(B) FBS 控制, 培养基辅以 10% FBS;(C) 血小板释放素-bb 控制, 4 ng/毫升的血小板生长因子-bb 在无血清培养基中;(D) 血小板-bb-bb (4 ng/毫升) 和抗血小板生长因子-bb (4 ng/毫升) 多克隆抗体在无血清培养基中;(E) 骨髓基质细胞组, 2 x 105骨髓基质细胞在无血清培养基中;和 (F) 骨髓基质细胞-p 组, 2 x 105骨髓基质干细胞在无血清培养基中。所有数据显示为平均± SEM (n = 4)。#: C 组和所有其他组 (F 组除外) 的比较, ## p & #60; 0.0001。*: F 组和所有其他组的比较, * p 和 #60; 0.05, *** 和 #60; 0.0001。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55381/55381fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" src="/files/ftp_upload/55381/55381fig4.jpg"> 
图 4: 高分辨率多显微镜 (倍/TPEF) 成像和定量的血管生成在小鼠颅骨缺损区。阮富仲 >> (a) 在 MPM 扫描下的鼠标示意图。橙色圆圈代表缺陷部位, 红色小管代表血管, 绿色代表胶原蛋白。(B) Unoperated 和操作动物被扫描与 MPM 在8周 post-implantation。倍, TPEF, 倍/TPEF 合并, 并显示3D 图像。虚线表示基于倍信号的控制群中骨骼缺陷的边界。(C) 在控制、PH 值和 PHp 的缺陷区域内量化的血管区域, 从2周到8周 post-implantation。所有数据显示为平均± SEM (n = 5)。: 组间 PH 值与 PHp、p 和 #60 的统计分析; 0.0001。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55381/55381fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" src="/files/ftp_upload/55381/55381fig5.jpg"> 
图 5:RT-qPCR 分析血小板生成因子-b (A) 和血管新生相关基因vWF (b) 和VEGRF2 (C) 表达式的三组。每个时间点对来自4只动物的样本进行了分析. #: PHp 和 PH 值组的比较, # p & #60; 0.05, ## p & #60; 0.01, ## p & #60; 0.0001. *: 比较 PHp 和对照组, * p & #60; 0.05, ** 和 #60; 0.01, 和 *** 和 #60; 0.0001。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55381/55381fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" src="/files/ftp_upload/55381/55381fig6.jpg"> 
图 6: 用 MicroCT 扫描评估体内骨形成。没有植入支架的控制组。仅在植入 PLGA/nHAp 支架内的 PH 值组。PHp 组内植入的 PLGA/nHAp 支架修改的 LV-pdgfb粒子。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55381/55381fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair at JoVE.com

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

在这里, 我们提出了一个协议, 以可视化血管形成在体内和在 real-time 的3D 支架的多显微镜。在小鼠颅骨临界骨缺损模型中研究了基因修饰支架的血管生成。治疗组发现更多的新血管。

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 ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

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

9.2K 次观看.  Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. 在这里, 我们提出了一个协议, 以可视化血管形成在体内和在 real-time 的3D 支架的多显微镜。在小鼠颅骨临界骨缺损模型中研究了基因修饰支架的血管生成。治疗组发现更多的新血管。

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

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

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

负染色的EM可视化蛋白质和大分子复合物:从电网的制备图像采集

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

视频率扫描共聚焦显微镜和内窥镜

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

评估白细胞内皮的相互作用下水流条件的<em&gt;离体</em&gt;自动注入微流室法

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

独特的毛细管作用通过微通道骨般的模板可以增强细胞的招募大骨缺损修复

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

CRISPR/Cas9 复合原注射液生产基因工程金黄叙利亚仓鼠

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...