项目受到Zentrales Innovationsprogramm的Mittelstand（ZIM）DES Bundesministeriums献给Wirtschaft UND科特布斯-KF3010902AJ4资助。出版费用已涵盖的BG创伤医院德国蒂宾根。

注:永生人类间充质基质细胞前体细胞（SCP-1细胞）用于实验。 SCP-1细胞通过的Matthias Schieker 12教授提供。 1. SCP-1细胞的扩展<ol><li>之前与SCP-1细胞的工作，正确清洁用70％乙醇（V / V）戴着手套工作区域（指定的生物安全柜Ⅰ）。 </li><li>在清洗过的生物安全柜中通过如表1所示混合所需组分制备细胞培养基的适当体积。为了维持培养基的无菌性，通过无菌过滤器传递具有0.22微米的孔尺寸添加补充剂。 <ol><li>为防止污染，使用前准备中至少24小时。为了测试其无菌性，孵育在细胞培养板1毫升培养基没有细胞中的标准细胞培养孵化器:37℃，5％CO 2，20％O 2和90％的湿度。后24小时，检查中使用显微镜的至少200倍的放大倍数。 </li></ol></li><li>保持SCP-1细胞与支持条件的标准的细胞培养孵化器:37℃，5％CO 2，20％O 2和90％的湿度。 </li><li>为了维护和扩展，成长SCP-1细胞，直到他们达到80-90％汇合。在这段时间内培养，培养基改变每2-3天。在达到80-90％汇合，拆分SCP-1的细胞（一般为1:2的比例）到下一个通道扩张或板用于实验（指示）。 </li><li>用于分离在SCP-1细胞，温培养基在37℃，使用水浴在37℃下解冻胰蛋白酶/ EDTA。 </li><li>完全吸从80-90％汇合细胞培养液，并弃置于垃圾容器。 </li><li>用DPBS洗涤细胞至少两次（Dulbecco氏磷酸盐缓冲盐水无镁和钙，pH值7.2）。 <ol><li>吸取适当体积的DPBS到细胞上（为一个T75培养瓶中5毫升DPBS和12ml的T175培养瓶）。 </li><li>吸仔细DPBS并丢弃到废物容器中。 </li></ol></li><li>吸取0.25％胰蛋白酶/ EDTA的适当体积到细胞上（1毫升一个T75培养瓶中的DPBS和2ml的T175培养瓶），并在具有5的标准细胞培养培养箱中于37℃孵育5-10分钟％的CO 2，20％O 2和90％的湿度。 </li><li>通过敲击容器逐出细胞并确保所有细胞从培养塑料（胰蛋白酶）通过在显微镜下观察漂浮细胞分离。 </li><li>灭活通过加入10ml培养基的胰蛋白酶反应。混合胰蛋白酶和细胞反复吹打仔细网上平台。 </li><li>细胞悬液转移入反应管中，离心分离，在600×g离心在室温下10分钟。 </li><li>吸出上清液，重悬细胞沉淀在10ml培养中。 </li><li>通过计数台盼蓝排除法的细胞在协议2描述。 </li><li>种子取决于实验设计的细胞。 </li></ol> 2. SCP-1细胞计数<ol><li>执行使用血球的再悬浮的细胞的活细胞计数（锥虫蓝排除法）。 </li><li>前的细胞数，用自来水清洗血细胞计数。干用无绒组织血球成分。濡玻璃盖成反比按下它到两个玻璃选手在计数区域的每一侧组装。确保牛顿环看到在玻璃板上跑步（ 图1）。 </li><li>采取10微升的再悬浮细胞，并用10微升的0.1％台盼蓝溶液混合，以获得2的稀释因子。 </li><li>负载10微升的预清洗和装配血细胞计数器室的总样本。 </li><li>计数活（白/透明细胞）和死亡人数（蓝色核）上的4×4平方细胞（参见图1B）。 </li><li>计算细胞的定式下列的总数: <img alt="图1" src="/files/ftp_upload/54537/54537eq1.jpg" /></li></ol> 3. SCP-1细胞的转染GFP 注意:为了观察并进入针织钛支架上我们标有绿色荧光蛋白（GFP）的细胞在一定培养期间SCP-1细胞的生长。 GFP的过表达是通过感染编码GFP的腺病毒颗粒来实现的。复制无能（-E1 / -E3）编码绿色荧光蛋白（GFP）的腺病毒颗粒用于感染SCP-1的细胞。通过收集重组腺病毒（Ad5的-GFP）转染HEK293T细胞（生物安全实验室II）的培养上清从史蒂芬·杜利教授13获得了病毒颗粒。三反复冷冻（-80℃）和解冻（37℃水浴）循环保证了没有，他K293T细胞保持存活，以产生新的病毒颗粒。使用这种腺病毒种子库可以有效地感染SCP-1细胞，而不会产生新的病毒颗粒。因此，感染的细胞可以在生物安全实验室一处理<ol><li>悬浮用的50,000个细胞/ ml的培养基中接种密度靶细胞（SCP-1细胞）。加入2毫升，每孔到6孔组织培养板。 </li><li>孵育在37℃下在标准细胞培养培养箱5％CO 2，20％O 2和90％湿度，直到SCP-1细胞达到70-80％铺满。 注:会合取决于细胞的接种密度。对于上述的条件时，SCP-1细胞达到70-80％在1.5-2天汇合。 </li><li>在70-80％铺满，吸不培养基每毫升培养液中加入100μl腺病毒种子库。 <ol><li>个别地取决于病毒颗粒在各病毒本身的量确定的浓度范围编辑备料。在情况下，感染效率太低，纯化和浓缩使用各种市售试剂盒的病毒颗粒。 </li></ol></li><li>在37℃下（5％CO 2，20％O 2和90％湿度）孵育在标准细胞培养孵化一小时。 </li><li>删除含有病毒种子储备培养基并将其收集处置。加入新鲜的培养液2毫升，每井的6孔培养板来补充的。 <ol><li>确保在处置之前，含中等病毒颗粒高压灭菌。 </li></ol></li><li>评估细胞内GFP表达（感染效率）使用带有GFP LED立方体/过滤器设置的荧光显微镜在感染后24小时。 <ol><li>观察细胞的形态（ 图2）。细胞从培养塑料分离可以给假阳性结果。 </li></ol></li></ol> 4.针织钛支架的清洁<ol><li> P花边高达5支架到50ml反应管。 </li><li>用30ml蒸馏水，去离子水洗净该支架（6-7毫米厚）三次，在室温下20分钟，使用转子条件（8 XG）。 </li><li>用30ml 1％的取代的蒸馏去离子水（重量/体积）的Triton-X-100溶液（溶解在蒸馏水去离子水），然后洗支架一次，在室温下20分钟，使用转子条件（8 XG） 。 </li><li>丢弃的Triton-X-100的溶液中，接着用30毫升蒸馏水 - 去离子水洗涤两次（每次5分钟，在室温下）保持转子条件（8 XG）。 </li><li>更换蒸馏水，去离子水。冲洗支架依次用试剂级99％丙酮，99％异丙醇和99％的乙醇（30ml每次）为2×5分钟的每个在超声浴（〜50赫兹，50瓦，220-240伏）。 </li><li>用30ml蒸馏水，去离子水再次洗涤三次5分钟，保持超声浴处理常数。 </li><li>放置scaffo为了在无绒布组织LDS至室温空气干燥过夜。 </li><li>用15磅高压灭菌该支架15分钟在121℃。 </li><li>作为协议的5所描述确认通过间接荧光清洗协议。 </li></ol> 5.成像支架结构由间接荧光备注:本协议通过使用荧光磺若丹明B上在586分之565纳米EX / EM波长给出了一个明亮的红色荧光间接免疫荧光支架描述结构的成像。但是，荧光团是可以改变的，以更好地适合于给定的显微镜设置或支架的可能自发荧光。 <ol><li>制备在1％乙酸和存储的磺酰罗丹明B染色溶液（0.04％），在室温下避光。 </li><li>取24孔组织培养板中，并通过将它INS沉浸于500μl的磺酰罗丹明B染色溶液的清洗支架IDE井使用镊子。 </li><li>捕获使用荧光显微镜支架的负像。 <ol><li>拍照与RFP LED立方体/滤波器40分之531纳米的激发波长和四十零分之五百九十三纳米的发射波长设置的。可替代地选择具有荧光光谱观察者20的帮助下适当激发和发射波长。磺酰罗丹明B在578纳米的高峰期激发和593纳米的峰值发射。 </li><li>为了形象化的支架结构，拍照在较低的放大倍率， 例如，4X或10X（ 图3）。使用这些图片，确定的特性， 例如，孔大小和与ImageJ的的帮助下成形。 </li><li>为了检测支架的杂质，拍照时的倍率， 例如，20X或40X，考虑到这会限制分析深度。确保灰尘颗粒/物质没有看到（见代表性的结果; 图URE 3）。 </li></ol></li></ol> 6. 在体外试验生物相容性<ol><li>在37℃的水浴中预加温培养基中。 </li><li>取24孔组织培养板和使用镊子，将清洗和消毒的支架中每个测试孔无菌。在预清洁生物安全柜我下工作！ </li><li>浸泡/孵化用培养基支架为约15分钟（每24孔组织培养板的孔500微升）中，为了从该支架的内部除去空气。 </li><li>与此同时，重悬500000的Ad-GFP感染的SCP1细胞于1ml培养基中。 </li><li>支架浸泡15分钟后，完全吸出介质关闭支架。 </li><li>用于接种细胞在支架上，免除小心在支架上（其被放置在24孔板）表面100微升细胞悬浮液。保持细胞悬浮液的最小体积，而接种的细胞，所以，以确保没有介质流ö支架的UT。 </li><li>孵育30分钟的细胞中，在37℃在标准细胞培养孵育箱（5％CO 2，20％O 2和90％湿度）。 </li><li>加入500μl更培养基并孵育在标准细胞培养孵化24小时（37℃，5％CO 2，20％O 2和90％湿度）。 </li><li>评估细胞附着图案和细胞使用荧光显微镜在支架表面上扩散。 <ol><li>拍照用GFP LED立方体/滤波器二十二分之四百七十零纳米的激发波长和四十二分之五百十纳米的发射波长设置的。或者选择适当的激发和发射波长的荧光光谱查看器的帮助。 GFP具有488nm的高峰期激发和507纳米的峰值发射。 </li><li>为了在较低的放大倍数， 例如，4倍或10倍（ 图4）可视化细胞粘附模式拍摄的照片。为了观察细胞扩散更高的MAGnification（至少100X）需要 - 限制了聚焦深度。 </li></ol></li><li>为进一步评估细胞在支架表面上铺展，固定用4％福尔马林的细胞并用细胞结构的传统的荧光染色， 例如，肌动蛋白丝（鬼笔环肽）进行。然而，适应的孵育时间和抗体的荧光标记，以适合个别脚手架特性， 例如，扩散时间或自体荧光14。 </li><li>次日，更换培养基以除去非粘附细胞。 </li><li>通过如在协议7所述刃天青转换测量计算在支架上的贴壁细胞的百分比。 </li></ol><img alt="图5" src="/files/ftp_upload/54537/54537fig5.jpg" /> 图5: 在体外试验时间安排 （ 一 ）电镀细胞的实验装置。 （B </st程序的荣&gt;）插图，强调直到第7天开始协议和细胞的功能验证的重要性， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 7.刃天青转换测量注:刃天青转化测定法用于测量线粒体活性，从而间接细胞增殖。刃天青还原成试卤灵产生荧光信号，这是基于与存活细胞数（ 图7A）相关的线粒体活性。 <ol><li>完全吸从SCP-1细胞的培养液中，并丢弃到废物容器中。 </li><li>用DPBS除去脱落的细胞冲洗SCP-1细胞一次。添加每个含在支架上的SCP-1细胞在24孔组织培养板的孔500微升DPBS。 </li><li>覆盖所需上午细胞无菌刃天青工作溶液（在培养基0.25％刃天青）和&#39;mount孵育在37℃下在标准细胞培养孵育箱（5％CO 2，20％O 2和90％湿度）中30分钟。 <ol><li>记下;孵育时间取决于细胞类型和细胞密度。它可以在10分钟和6小时之间变化。优化的温育时间为:SCP-1细胞是30分钟。 </li></ol></li><li>作为背景对照，包括至少一个孔与刃天青工作溶液，但没有细胞，即在37℃下在标准细胞培养培养箱中培养（5％CO 2，20％O 2和90％湿度）为同一多少时间。 </li><li>转移100μl的空调上清液从24孔组织培养板的各孔于96微孔板。 </li><li>用1毫升DPBS在室温下5分钟洗涤剩余的细胞三次，以去除残留的刃天青工作溶液。第三次洗涤后添加培养基Ť直径:与SCP-1细胞的植入物（每24孔组织培养板的孔500微升），并继续在37℃下温育在标准细胞培养孵育箱（5％CO 2，20％O 2和90％湿度）进一步的时间过程的测量。 <ol><li>确保在该步骤（2-4），以尽量减少移液误差来设置重复。 </li></ol></li><li>在此期间，将96微孔板入酶标仪并测量形成的试卤灵的荧光。 <ol><li>为了降低背景信号，在545纳米的激发波长和585nm的发射波长测量荧光。 </li><li>或者选择适当的激发和发射波长的荧光光谱查看器的帮助。试卤灵具有572纳米的高峰期激发和585纳米的峰值发射。给定的信号强度是25个别读数（每孔25闪烁）的平均值。 </li></ol></li><li>测量氟在条件培养基形成试卤灵的escence，使用底部光学器件。 <ol><li>作出这样的，增益取决于平均量刃天青转换并可以在10和4000之间变化的说明。调整吹打一个试卤灵的标准曲线，以便该荧光信号是低于最大信号强度可检测的80％（在此情况下，20,000）所使用的增益到各个酶标仪。对于SCP-1细胞的优化增益为800。 </li></ol></li><li>减去背景信号（刃天青工作液没有细胞）从所述测试样品的信号。 </li><li>根据实验装置/宗旨，继续进一步的步骤: </li><li>计算上使用的标准曲线的支架的细胞的生存力的百分比。分别使个体的标准曲线对所使用的每个细胞系。 </li><li>通过估算刃天青转换整个培养时间相对增加分析细胞生长/增殖。对于这个计算，设置作为基准1天刃天青转换。刚准备对这种分析的刃天青工作溶液。此外，孵育时间必须是不同的测量之间相等。 </li><li>重复刃天青转换测量的整个协议至少三次，以得到一致的结果。 </li></ol> 8.活死细胞染色<ol><li>板与50,000个细胞/支架的接种密度在支架的SCP1细胞，并允许它在支架上保持的标准细胞培养条件下生长（参见方案1和2）。 </li><li> 24-48小时后完全吸从SCP-1细胞的培养液中，并丢弃到废物容器中。 </li><li>用DPBS除去脱落的细胞冲洗SCP-1细胞一次。加入500微升DPBS每24孔组织培养板的孔，并孵育在室温下5分钟。 </li><li>染色SCP-1用荧光团（同时所有三个污渍）: <ol><li>从现在开始保持在黑暗中的支架，以防止荧光漂白从白天！ </li><li>设定的温育时间为30分钟，以允许在整个支架相等分布。如果转移到其他的支架，以确保最优化的温育时间，以适应个别脚手架特性（孔径，脚手架深度等 ）。 </li><li>为了检测在支架上的活细胞，染色用钙黄绿素AM的细胞以2μM的终浓度（在培养基中）。 </li><li>为了检测在支架上的所有细胞，染色用Hoechst 33342细胞在0.002微克/微升（在培养基中）的终浓度。 </li><li>为了检测死细胞，孵育乙锭同二聚体，支架在4微米（在培养基中）的最终浓度。 </li></ol></li><li>温育时间之后，洗涤细胞用DPBS 3次（1毫升，每孔）在室温下，每次5分钟。 </li><li>立即拍照S按使用荧光显微镜。 <ol><li> （分别为二十二分之四百七十零nm和四十二分之五百十纳米，激发和发射波长）取钙黄绿素的照片（活细胞）用GFP LED立方体/过滤器集。或者选择一个适当的激发和发射波长的荧光光谱查看器的帮助。钙黄绿素具有488nm的高峰期激发和507纳米的峰值发射。注:确保不使用GFP转染细胞与钙黄绿素或其他绿色荧光染料荧光染色。 </li><li>取的Hoechst 33342的照片使用DAPI LED立方体/滤波器44分之357纳米的激发波长和六十零分之四百四十七纳米的发射波长设置的。或者选择适当的激发和发射波长的荧光光谱查看器的帮助。赫斯特33342具有347纳米的高峰期激发和483纳米的峰值发射。 </li><li>就拿乙锭同型二聚体的照片与RFP LED立方体/过滤器设置（激发和发射波长Ø˚F四十零分之五百三十一nm和四十零分之五百九十三纳米，分别）。或者选择适当的激发和发射波长的荧光光谱查看器的帮助。乙锭同型二聚体具有530nm的高峰期激发和618纳米的峰值发射。 </li></ol></li></ol>

<ol>
	<li>Bridwell, K. H., Anderson, P. A., Boden, S. D., Vaccaro, A. R., Wang, 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=What's+new+in+spine+surgery.">What's new in spine surgery.</a> J Bone Joint Surg Am. 95, 1144-1150 (2013).</li><li>Adams, M. A., Dolan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+degeneration:+evidence+for+two+distinct+phenotypes.">Intervertebral disc degeneration: evidence for two distinct phenotypes.</a> J Anat. 221, 497-506 (2012).</li><li>Schizas, C., Kulik, G., Kosmopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disc+degeneration:+current+surgical+options.">Disc degeneration: current surgical options.</a> Eur Cell Mater. 20, 306-315 (2010).</li><li>Lewis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleus+pulposus+replacement+and+regeneration/repair+technologies:+present+status+and+future+prospects.">Nucleus pulposus replacement and regeneration/repair technologies: present status and future prospects.</a> J Biomed Mater Res B Appl Biomater. 100, 1702-1720 (2012).</li><li>Cunningham, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+scientific+considerations+in+total+disc+arthroplasty.">Basic scientific considerations in total disc arthroplasty.</a> Spine J. 4, 219-230 (2004).</li><li>Buck, A. E., Kaps, H. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implant+for+surgical+use+in+humans+or+vertebrates.">Implant for surgical use in humans or vertebrates.</a> US8728164 B2. Google Patents. , (2014).</li><li>Kettler, A., Kaps, H. P., Haegele, B., Wilke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+behavior+of+a+new+nucleus+prosthesis+made+of+knitted+titanium+filaments.">Biomechanical behavior of a new nucleus prosthesis made of knitted titanium filaments.</a> SAS J. 1, 125-130 (2007).</li><li>Nerurkar, N. L., Elliott, D. M., Mauck, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+design+criteria+for+intervertebral+disc+tissue+engineering.">Mechanical design criteria for intervertebral disc tissue engineering.</a> J Biomech. 43, 1017-1030 (2010).</li><li>Elias, C. N., Lima, J. H. C., Valiev, R., Meyers, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+titanium+and+its+alloys.">Biomedical applications of titanium and its alloys.</a> JOM. 60, 46-49 (2008).</li><li>Hallab, N., Link, H. D., McAfee, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+optimization+in+total+disc+arthroplasty.">Biomaterial optimization in total disc arthroplasty.</a> Spine (Phila Pa 1976). 28, 139-152 (2003).</li><li>Gustafsdottir, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+cytological+profiling+assay+to+measure+diverse+cellular+states.">Multiplex cytological profiling assay to measure diverse cellular states.</a> PLoS One. 8, e80999 (2013).</li><li>Bocker, W., 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=Introducing+a+single-cell-derived+human+mesenchymal+stem+cell+line+expressing+hTERT+after+lentiviral+gene+transfer.">Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer.</a> J Cell Mol Med. 12, 1347-1359 (2008).</li><li>Ehnert, 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=Transforming+growth+factor+beta1+inhibits+bone+morphogenic+protein+(BMP)-2+and+BMP-7+signaling+via+upregulation+of+Ski-related+novel+protein+N+(SnoN):+possible+mechanism+for+the+failure+of+BMP+therapy.">Transforming growth factor beta1 inhibits bone morphogenic protein (BMP)-2 and BMP-7 signaling via upregulation of Ski-related novel protein N (SnoN): possible mechanism for the failure of BMP therapy.</a> BMC Med. 10, 101 (2012).</li><li>Morgan, S. P., Rose, F. R., Matcher, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optical Techniques in Regenerative Medicine. , (2013).</li><li>Vielreicher, 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=Taking+a+deep+look:+modern+microscopy+technologies+to+optimize+the+design+and+functionality+of+biocompatible+scaffolds+for+tissue+engineering+in+regenerative+medicine.">Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine.</a> J R Soc Interface. 10, 20130263 (2013).</li><li>Curtis, A., Wilkinson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographical+control+of+cells.">Topographical control of cells.</a> Biomaterials. 18, 1573-1583 (1997).</li><li>Niu, G., 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=Fluorescent+imaging+of+endothelial+cells+in+bioengineered+blood+vessels:+the+impact+of+crosslinking+of+the+scaffold.">Fluorescent imaging of endothelial cells in bioengineered blood vessels: the impact of crosslinking of the scaffold.</a> J Tissue Eng Regen Med. , (2014).</li><li>Chan, B. P., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolding+in+tissue+engineering:+general+approaches+and+tissue-specific+considerations.">Scaffolding in tissue engineering: general approaches and tissue-specific considerations.</a> Eur Spine J. 17, 467-479 (2008).</li><li>Navarro, M., Michiardi, A., Castano, O., Planell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+in+orthopaedics.">Biomaterials in orthopaedics.</a> J R Soc Interface. 5, 1137-1158 (2008).</li><li>Priyadarshani, P., Li, Y., Yao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+biological+therapy+for+nucleus+pulposus+regeneration.">Advances in biological therapy for nucleus pulposus regeneration.</a> Osteoarthritis Cartilage. , (2015).</li><li>. Thermofisher Fluorescence Spectraviewer Available from: <a target="_blank" href="https://www.thermofisher.com/de/de/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html">https://www.thermofisher.com/de/de/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html</a> (2016)</li></ol>

作者宣称他们没有利益冲突。工作的任何部分已经或正在考虑公布或已在其他地方发表。

在支架表面起着其相互作用与体内周围组织，从而确定植入官能耐久性重要的作用。因此，支架的生物相容性，通过使用细胞（SCP1细胞系） 的体外测定，对支架镀时的研究。 与薄和光学透明支架功能良好的显微技术适合较差的非透明支架研究生物相容性。这主要是由于非透明支架防止未经显著失真15穿透光。为了部分解决这些问题，我们在此建立在/在使用各种荧光针织钛制成的支架细胞评价的方法。 为了使编织接着钛导线折叠，材料抵接强腐蚀性和有毒物质（润滑剂，媒染剂，电-polishing溶液）中，如果迹线保持在/脚手架上可能改变支架的生物相容性。随着开发间接荧光协议的帮助（协议5），我们可以想像的支架结构。此外，脚手架特性， 例如 ，材料的厚度，各个孔的尺寸和形状，或结缔组织密度，使用ImageJ进行分析。较高放大萤光显微镜图像中允许的支架以及所述支架表面上的可视化的杂质。 图3b从而表示成功开发清洁协议的验证结果。这种间接染色方案的原理可以很容易地再生到其他非透明的支架，考虑到个人的支架属性， 例如 ，孔径大小和对应的扩散其影响的温育时间。而且，该支架和微观设置自动荧光影响FL的选择使用uorophores。在线工具荧光光谱查看器可以帮助选择适当的荧光。 细胞 - 金属相互作用已由在支架上分析细胞的粘附图案间接检测。协议6描述了如何细胞可以在体外 ，如果通过使用GFP的转染策略镀上在培养系统非透明支架进行监测的方法。基于在体外测定的初步结果，已经预测，如组合物中，微地形和粗糙度16的表面特性可能在建立粘附和靶细胞的扩散中起重要作用。钛被生物材料从而可能被用作衬底模板，以提供细胞附着（参照图4）基。 植入物的表面形貌已经报道了影响细胞的行为16。在本研究中，我们分析了细胞生长/扩展和使用活力荧光染色技术结合定量生存力测量。分析显示，在细胞百分比生存力微妙变化，并根据该脚手架材料蔓延。然而，通过刃天青转化测定（线粒体活性）定量评估细胞的功能，并没有显著由该脚手架材料的影响。通过刃天青转换线粒体活性的测量有它不是细胞毒性的优点，因此可以反复在长时间培养期间进行。特别注意有掉残余刃天青工作溶液洗涤时，所以不会积聚背景信号（假阳性结果）才能作出。尽管有这些优点，刃天青转化测定不会给对细胞在支架上传播的任何信息。 GFP的感染细胞，因此可以在很长的培养期（GFP信号仍然是14天不变）进行跟踪，从而使可视化小号支架表面上pecific增长方式的转变。细胞在支架更深的可视化仍然由支架材料的限制，因此将需要植入物的清扫。这两种技术的组合具有的主要优点在于它可以很容易地转移到其他的细胞类型，考虑到孵育时间可能必须适于感兴趣的细胞类型。然而，护理有转移这种方法与其他非透明支架， 例如 ，当将要采取，基于胶原蛋白通常表现出强烈的绿色自体荧光支架17。在这种情况下，可能会使用其它荧光标记。因此，上述两种方法的组合有几个优点（ 见表2）。 脚手架特性， 例如 ，孔支架内大小可能陷阱细胞。如果没有足够的营养供给，这些细胞可能会死，分泌影响环绕的细胞活性蛋白酶ING细胞/组织。我们能够适应基于荧光染色协议，是能够可视化活的和死的细胞中和在针织钛支架。类似于间接荧光染色方案，此染色方案的原理可以很容易地转移到其他非透明支架。而这样做时，个人的支架属性， 例如 ，孔径大小和相应的扩散以及可能的自体荧光，显微设定必须考虑到，因为它们可能会影响培养时间和荧光团的使用的选择。在这里，再次的在线工具荧光光谱查看器可以帮助选择适当的荧光。 总之， 在体外结果表明，所提出的针织钛核植入物模型具有生物分布。本材料间充质前体细胞（SCP-1细胞）的初始附着表明，这种钛合金种植体本草l为生物相容性。虽然我们还没有分析了不同地形参数的关联，支架表面修饰可以增强细胞粘附增殖和分化18。支架和细胞之间的最佳兼容性提高更好的植入物的整合的概率到周围组织和处理19后，以便提高在体内的寿命。使用上述测试装置开辟了测量，并在通过表面修饰诱导支架的生物性能可视化的改进的可能性。支架的生物活性表面上未修改的植入物的设计偏好表明在骨，软骨整合方面更好的性能20。这项研究是由哪里的力学性能和生物活性已有报道6,7针织钛种植体其他报告进一步增强。

慢性背部疼痛是一种多因素疾病。自20世纪50年代的椎间盘退行性疾病的微创治疗选择的兴趣增加。直到今天，在脊柱的多节段的融合是最广泛使用的治疗方法。因为，这种方法常常导致在受影响的段1,2的移动性限制，关节造形术时代的勘探成为广泛的兴趣。总椎间盘置换髓核置换显著进步已成为治疗慢性背痛1一个很好的选择。尽管巨大的进步，没有一种方法已被临床评估。少刚性核植入物代表一个有前途的替代全椎间盘置换，只要该纤维环是完好3,4。然而，市场上的当前存在的核植入物通常伴有并发症像在椎体，错位，盘和叔垂直高度损耗变化他缺乏必要的相关机械刚性5。为了克服目前的缺点，提出针织钛线的新型核植入物已研制成功6。由于独特的编织结构，这种新开发的脚手架已经显示出杰出的生物力学性能， 如阻尼特性，孔径，承载能力和可靠性7。在归因于植入物的非透明性的（光学）分析技术旨在测试此新颖核植入物的生物相容性，描绘严重限制。 为了测试生物相容性，细胞-金属相互作用中起着突出的作用8-10。细胞和支架之间的相互作用是必要的稳定，从而为在主机系统内的更好的植入物的整合。然而，越来越长入深度可能改变支架的机械性能。旨在不变拟tigate所述支架表面是否提供了用于细胞附着，增殖和分化，或碱金属是否影响细胞生存力，它解决在非透明和不透明支架成像上/细胞的常见公知的问题是重要的。为了克服此限制几个荧光灯为基础的技术进行了探讨。公司提供大范围的荧光的可视化活细胞，细胞器，甚至是特定的细胞状态11。此实验的荧光团进行，以最好地适合我们的荧光显微镜与在线工具光谱观看者的帮助​​下选择。 上/在非透明针织钛支架贴壁细胞行为的分析开发策略涉及以下内容:osteochondro祖细胞的1）的荧光（绿色荧光蛋白/ GFP）标记，以允许在细胞的跟踪脚手架，2）测定存活率（线粒体chondrial活性）的细胞，以及3）可视化的细胞 - 细胞和所述支架内的细胞物质的相互作用。该过程具有的优点在于它可以很容易地转移到其它贴壁细胞和其他非透明或不透明的支架。此外，生存力和向内生长模式可以在几天进行监控，因此，它可以用有限数量的支架材料或细胞的使用。 本研究证明的成功使用我们的当前协议来测量细胞活力和可视化在生长上/ osteochondro祖细胞的图案在非透明针织钛支架。此外，开发的协议可能，以便确定所述支架的杂质，并检查清洗协议一起使用。

<table><tbody><tr><td>6/24/48 well plates, T25/ T75 culture flask</td><td>Greiner Bio-One GmbH</td><td>*</td><td></td></tr><tr><td>* 24 well plates</td><td>Greiner Bio-One GmbH</td><td>CELLSTAR 662 160</td><td></td></tr><tr><td>* 48 well plates</td><td>Corning Incorporated USA</td><td>3548</td><td></td></tr><tr><td>* 6 well plates</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>* T25</td><td>Greiner Bio-One GmbH</td><td>690 175</td><td></td></tr><tr><td>* T75</td><td>Greiner Bio-One GmbH</td><td>658 175</td><td></td></tr><tr><td>Acetic acid, purum &amp;ge; 99.0%</td><td>Carl Roth</td><td>3738.4</td><td></td></tr><tr><td>Acetone</td><td>Carl Roth</td><td>5025.1</td><td></td></tr><tr><td>Axioplan-2&amp;nbsp;</td><td>Carl Zeiss, Germany</td><td></td><td></td></tr><tr><td>Biological safety cabinets</td><td>Thermo Scientific</td><td>safe 2020</td><td></td></tr><tr><td>Calcein acetoxymethyl ester (calcein AM)</td><td>Sigma</td><td>17783</td><td></td></tr><tr><td>Cell Culture Incubtator</td><td>Binder, Tuttlingen, Germany</td><td>9040-0078</td><td></td></tr><tr><td>Filter unit (0.22 &amp;micro;m)</td><td>Millipore, IRL</td><td>SLGP033RS</td><td></td></tr><tr><td>Centrifuges 5810 R And 5417 R</td><td>Thermo Fisher Scientific, NY</td><td>Megafuge 40R</td><td></td></tr><tr><td>Dimethylsulfoxid (DMSO)</td><td>Carl Roth</td><td>4720.2</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s PBS without Ca &amp;amp; Mg</td><td>Sigma</td><td>H15-002</td><td></td></tr><tr><td>Ethanol 99%</td><td>&amp;nbsp;SAV liquid prod. GmBH</td><td>475956</td><td></td></tr><tr><td>Ethidium homodimer</td><td>Sigma</td><td>46043</td><td></td></tr><tr><td>EVOS Fluorescence imaging system</td><td>Life technologies</td><td>AMF4300</td><td></td></tr><tr><td>Fetal Bovine Serum (FCS)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific, PA, USA</td><td></td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma</td><td>14533-100MG</td><td></td></tr><tr><td>Knitted titanium nucleus implant</td><td>Buck co &amp;amp; KG,Germany</td><td></td><td></td></tr><tr><td>MEM Alpha Modification with Glutamine w/o nucleoside</td><td>Sigma</td><td>E15-832</td><td></td></tr><tr><td>Omega microplate Reader</td><td>BMG Labtech,Germany</td><td>FLUOstar Omega</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma</td><td>P11-010</td><td></td></tr><tr><td>Resazurin sodium salt</td><td>Sigma</td><td>199303-1G</td><td></td></tr><tr><td>Sulforhodamine B sodium salt</td><td>Sigma</td><td>S1402-1G</td><td></td></tr><tr><td>Test tube rotator</td><td>Labinco B.V.,The Netherlands</td><td>Model LD-76</td><td></td></tr><tr><td>TRIS (hydroxymethyl) aminomethan</td><td>Carl Roth</td><td>AE15.1</td><td></td></tr><tr><td>Triton</td><td>Carl Roth</td><td>3051.2</td><td></td></tr><tr><td>Trypan Blue 0.5%</td><td>Carl Roth</td><td>CN76.1</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Sigma</td><td>L11-004</td><td></td></tr></tbody></table>

imaging cell viability on non-transparent scaffolds &#8212; using the example of a novel knitted titanium implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

初步结果表明，所描述的新颖的核植入物不仅具有良好的阻尼特性，而且是生物相容与SCP-1的细胞。在植入物的生产过程中，它与强腐蚀性和有毒物质（润滑剂，媒染剂，电解抛光溶液）接触。用间接荧光染色技术的帮助下，我们能够可视化剩余的杂质，从而优化示出在支架上物质负载显著减少清洁协议; 图3示出了建立的清洁协议的效率。 用于关节成形术治疗是由在蜂窝材料的界面发生的事件来确定植入物的成功与否。 图4示出了附连在支架后电镀的24小时的细胞，如在协议部分6，一种显著反式描述观察到SCP-1细胞的染效率，因为我们可以像在支架上（参照图2）间充质基质细胞前体细胞的生长分析。直接可视确认支架的生物相容性和也示出了支架表面上的附着图案（ 图4）。荧光染色可以做进一步的检查与和支架表面上散布细胞相互作用。 荧光团，以检查细胞死亡和增殖过度在支架的一段时间已成功应用。活死染色的图像举例说明如何染色可以成功地在支架上，以确认细胞在一段时间的百分数可行性来完成。 图6示出了蓝色的核染色（Hoechst的3342）中的所有细胞中，红色荧光标记的（乙锭同型二聚体）死细胞，以及钙荧光素AM掺入作为可行性马环保标签rker。钙黄绿素AM被转化为钙黄绿素其显示在钙离子在细胞的细胞质中的存在的明亮的绿色荧光。赫斯特33342是细胞壁可渗透和插层到细胞的DNA。这样，所有细胞将显示蓝色的细胞核（参照图6）。乙锭同型二聚体是不细胞壁可渗透的，因此，它只会地嵌入到死细胞的DNA中。这样一来，死细胞将显示红色的细胞核。此外，细胞存活率和折叠每周增加细胞数量上支架被刃天青转化测定定量和用图形表示（ 图7）。 <img alt="图1" src="/files/ftp_upload/54537/54537fig1.jpg" /> 图1: 细胞用血细胞计数器计数 （A）中的腔室组件的安装。 （B）的计数板插图; 4×4计数室被用于细胞凑NT。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54537/54537fig2.jpg" /> 图2:绿色荧光蛋白转染效率 SCP1细胞表现出强的绿色荧光指示正广告GFP-转染效率。比例尺= 1000微米，放大4倍。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54537/54537fig3.jpg" /> 图3: 磺酰罗丹明B染色阴性的图像捕捉清洗前（A）脚手架。箭头指示的有毒/腐蚀性物质对支架的存在。 （B）清洗后的协议脚手架。比例尺= 1000微米，放大4倍。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54537/54537fig4.jpg" /> 图 4: 在脚手架 GFP信号SCP1细胞的黏附模式表示针织钛支架表面上的细胞粘附和增长方式的转变。比例尺= 1000微米，放大4倍。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54537/54537fig6.jpg" /> 图 6: 支架细胞联合荧光染色（。 <st荣&gt; A）的赫斯特核染色（蓝色）和（B）的钙荧光素-AM胞浆染色（绿色）。 （C）的箭头表示死细胞的存在下，由于乙锭同型二聚体-1染色（红色）的摄取。 （D）示出了合并的图像。比例尺= 1000微米，放大4倍。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54537/54537fig7.jpg" /> 图7: 刃天青转化测定 （A）氧化还原染料（刃天青）的生化还原反应到最终产品（试卤灵）发出荧光和色度发生变化。 （B）当细胞在0.75毫克/立方厘米密度的支架铺板测定线粒体活性。数据使用FL收集基于荧光测量仪。在在定义的时间点（X轴）590纳米（y轴）荧光强度被描绘为定量细胞生存力测定（实施例= 540纳米，EM = 590纳米）的结果。采用双向ANOVA和平均值的标准误差（SEM）示出作为误差棒被确定统计显着性。考虑到支架的物理特性， 例如，孔径大小和力学性能（如减震功能）一起，被用于表征生物相容性0.75毫克/立方厘米密度的支架。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 媒体组件 </td><td> 浓度 </td></tr><tr><td>基底αMEM培养基（％） </td><td> 90 </td></tr><tr><td>血清（％） </td><td> 10 </td></tr><tr><td>笔/链球菌（％） </td><td> 1 </td></tr></tbody></table> 表1:细胞培养（αMEM）媒体组合物。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 问题 </td><td> 原因 </td><td> 解 </td></tr><tr><td>非透明脚手架细胞活力成像</td><td>脚手架防止无失真光穿透</td><td>使用基于荧光成像技术进行细胞的评估。 </td></tr><tr><td>荧光强度的干扰</td><td>自动荧光，背景信号</td><td>需要注意的是荧光团，并根据特定支架性能使用为宜。 </td></tr><tr><td>脚手架上通过培养期细胞可行性分析</td><td> - [R过了很久epeated测量</td><td>转用Ad-GFP-病毒颗粒细胞。 </td></tr><tr><td>在非透明的支架细胞功能评估</td><td>荧光成像技术，只允许传播模式分析单元。 </td><td>在与成像技术的组合进行刃天青转化测定（定量细胞活力测量）。 </td></tr></tbody></table> 表2:汇总表:非透明的支架故障排除细胞活力成像。

Watch this Scientific Journal Video about Imaging Cell Viability on Non-transparent Scaffolds â€” Using the Example of a Novel Knitted Titanium Implant at JoVE.com

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

这里，我们提出一个基于荧光的成像技术来检测细胞生存力上的非透明钛脚手架以及检测的脚手架杂质影子。这个协议进行故障排除在非透明支架成像细胞 - 细胞或细胞 - 金属相互作用的缺点。

非透明的支架成像细胞活性 - 使用新型针织钛种植体为例

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

imaging-cell-viability-on-non-transparent-scaffolds-using-example

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

11.3K 次观看.  Eberhard Karls Universität Tübingen. 这里，我们提出一个基于荧光的成像技术来检测细胞生存力上的非透明钛脚手架以及检测的脚手架杂质影子。这个协议进行故障排除在非透明支架成像细胞 - 细胞或细胞 - 金属相互作用的缺点。 

视频: Imaging Cell Viability on Non-transparent Scaffolds — Using the Example of a Novel Knitted Titanium Implant

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

科研

生物工程

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

多尺度孔梯度，聚电解质和间接监测改性金属植入物<em&gt;在体内</em

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

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 ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

癌症研究

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 ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

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

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

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

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

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

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

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

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

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

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

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

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

非透明的支架成像细胞活性 - 使用新型针织钛种植体为例

摘要

探索更多视频

此视频中的章节

静电纤维组织工程的后期处理

多尺度孔梯度，聚电解质和间接监测改性金属植入物

一个完整的皮肤缺损模型，以评估生物材料的血管化

手术技术组织工程血管移植和后续的植入

使用胶原基支架的神经母细胞瘤三维体外仿生模型

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

加工机械可调和生物活性金属支架的生物医学应用

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

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

管状结构组织工程的核心/壳印刷支架

非透明的支架成像细胞活性 - 使用新型针织钛种植体为例

摘要

探索更多视频

此视频中的章节

静电纤维组织工程的后期处理

多尺度孔梯度，聚电解质和间接监测改性金属植入物

一个完整的皮肤缺损模型，以评估生物材料的血管化

手术技术组织工程血管移植和后续的植入

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

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

加工机械可调和生物活性金属支架的生物医学应用

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

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

管状结构组织工程的核心/壳印刷支架

使用胶原基支架的神经母细胞瘤三维体外仿生模型