作者想感谢安德烈沃尔德博士和尼古拉斯·佩兰制造钛种植体，K. Benmlih为积聚的聚四氟乙烯模具和G.普雷沃博士对他的帮助与动物实验。我们也承认本地区阿尔萨斯和PMNA的（极MATERIAUX等纳米科学阿尔萨斯）的财务贡献。

1。在孔的金属植入物的制备微孔梯度<ol><li>清洁植入物（如医用级钛有孔玻璃珠的大小范围为400-500微米，法国Neyco SAS，制成的植入物）的乙醇中，然后在丙酮中超声处理15分钟。 </li><li>根据植入物的大小和形状（对于标准的实验中，使用2厘米高度1.5厘米直径的圆筒状的模具）的设计和制造聚四氟乙烯模具。模具应该是模块化的，因此在提取过程中的某些部分可以被删除。这种模具的设计确保在提取过程中的控制，通过强制的提取液中的运动以定向的方式。 </li><li>管形植入物的设计，以取代气管的结构应该是由三件：ⅰ）的底部部分的大小来决定的植入物，ⅱ）的心轴以及iii）一个外芯，这是可移动的。通过这种方式，可以形成孔径梯度从外部向内侧。 </li> &lt;LI&gt;准备合成聚合物（PLLA）解决方案：对于需要准备冻结/提取聚乳酸溶液在二恶烷和水（87:13，V：V）的二元混合物。 </li><li>把混合物加热到60℃，以便获得均匀的溶液。 60℃的选择，因为它是为精密玻璃注射器的溶液引入到植入物已被使用的耐温性更高的极限。 </li><li>如果高浓度（≥6％），高分子量的聚乳酸的解决方案的使用，引入该溶液，立即加热后进入植入物。否则凝胶溶液发生不充分浸泡。 </li><li>计算所需的聚合物溶液的体积相对于植入物的孔隙度，在冷冻溶液的体积的准确度变化可以被考虑在内。 </li><li>到0.1μL精度的精密玻璃注射器植入介绍的解决方案。的聚合物浓度的下限值是孔梯度形成重复性的3％，而变得难以得到均匀分布在6％以上的厚的样品。然而，对于特定的应用程序，可以使用其它浓度。 </li><li>冻结的样品可直接在-80℃或30分钟，在室温下与现有的潜伏期。冷冻条件确定部分的孔形成的，因而冷冻的条件下可以进行调整的孔隙率，旨在保持在-80℃下的样品过夜</li><li>提取：80％预冷的乙醇浸泡植入。在-20°C进行提取过夜。要获得孔隙率梯度，删除所有模具零件的管状植入物和所有的部件，除了底部圆盘形植入芯棒除外。使用预冷的手术刀的模具，以方便分离。 </li><li>萃取后在-20℃下保温过夜19，除去剩余的模具部件和空气干燥的植入物。为表征的的结构压汞仪分析的整体的孔隙率是必要的。汞孔度计测量表现出明显的峰值对应的植入物和的小interdispersed的毛孔的两面上的孔。然而，是较为重要的数据是腔内表面和腔外表面，可以分析图像J的孔径分布用扫描电子显微镜（SEM）20的孔隙率之间的差异。为了核实孔梯度，冷冻断裂样品，并用SEM观察横截面。 </li><li>由于开放的多孔性质，所用的植入物，和乙酸的光反射能力，这是可以做到的z轴堆栈的标记细胞在多孔植入物。标记细胞与PKH26或钙黄绿素-AM和可视化的激光共聚焦显微镜植入。 </li></ol> 2。表面涂层与胶原/海藻酸钠多层的多孔金属植入物<ol><li>对于建立多层，最高的可重复性浸渍机器人。但是，如果浸渍机器人，不提供这些步骤可以手动完成。 </li><li>使用医疗级胶原蛋白I型和海藻酸钠。的优化浓度0.5 g / L的150 mM氯化钠在pH 3.8的柠檬酸盐缓冲液中的每个。 </li><li>溶解胶原溶液过夜，以确保溶液的均匀性。为3.8的酸性pH值是必要的稳定的积聚层，在中性pH条件下的交联前的结构是不稳定的。 </li><li>植入胶原蛋白和海藻酸钠溶液交替浸泡，浸渍机器人系统的沉积层。每个后续层沉积时间为15分钟。冲洗5分钟用150 mM氯化钠在pH 3.8的沉积步骤之间的结构。 </li><li>设计特定的人利用植入物浸渍聚电解质多层生产中使用的机器人。沉积层，无论是钛的表面只有IMPLANTS或植入修改，如第1节所述。 </li><li>基底膜的镇定模仿与京尼平：准备交联溶液中的二甲亚砜（DMSO）/柠檬酸盐缓冲液（150mM的氯化钠，pH值3.8）在1:4 v：V比。宽的浓度范围内，可以使用，和100mM是足够的交联。溶解京尼平首先在二甲基亚砜组件，稍后加水成分，以避免结块。 </li><li>交联样品的交联溶液中浸泡12-24小时之间。之后用清水冲洗以大量的柠檬酸缓冲液（pH值3.8）。 </li><li>洗涤步骤后，使用UV处理（30分钟）或抗生素/抗真菌浴（青霉素/链霉素，两性霉素B）灭菌样品。 </li><li>确定基底膜模仿的质量的主要参数是它的厚度和纤维的直径。计算纤维直径，利用原子力显微镜（AFM）在接触模式下获得的图像。干燥样品的成像前的氮气流量。量化的每幅图像中的至少10根纤维的厚度，以确定原纤维的平均厚度的Image J软件。 </li><li>薄膜的厚度可以通过原子力显微镜（AFM）的划痕试验。干（COL / ALG）24 / COL多层的薄膜。用注射器针头划伤膜。经过本地化的划伤用光学显微镜，获得10×10微米的表面划痕的边界与AFM图像。计算的高度，用AFM软件，它提供的薄膜层的厚度得到的档案。 </li></ol> 3。 在体内植入集成间接监测血浆分析<ol><li>所有的，应当采取必要的委员会的批准用于动物实验，根据每个国家的21对管限规则。在我们的情况下，指南的保养和使用实验动物（国家研究委员会，2010）其次是和日ê斯特拉斯堡大学伦理委员会的批准。 </li><li>在目标站点植入。被用于新西兰大白兔15毫米气管切除气管置换协议，这里给出的血液监测。 </li><li>植入后每天随访是必要的，如监控中一般动物的福祉（愈合的手术部位，呼吸速率），并记录他们的体重左右。 </li><li>为了验证血液测试，使用一套行之有效的方法，如酶联免疫吸附试验血CRP水平。许多动物的CRP测试可用于兔特定的测试列于表1。同样，使用免疫印迹CGA水平的决心。这个协议被用于单克隆抗的CGA抗体（抗-CGA 47-68）。 </li><li>等离子体特性，获得兔耳缘静脉血液样本。在5,000 rpm离心20分钟，在4°; C.使用得到的上清液进行分析。在我们的程序中，这些测试是每周的基础上完成的，但也可以更频繁的测试。 </li><li>反相HPLC纯化的血浆蛋白含量：提取兔血浆中，用0.1％三氟乙酸（1:1，体积比）。纯化的提取物，通过在Dionex HPLC系统（终极3000;美国加利福尼亚州森尼韦尔）上的的反向NUCLEOSIL相300-5C18柱（4×250毫米，颗粒大小为5μm;孔隙度，300）。 </li><li>记录在214和280 nm处的吸光度。所用的溶剂体系是ⅰ）溶剂A：0.1％（体积/体积）三氟乙酸（TFA）在水和ii）溶剂B：0.09％（体积/体积）TFA的70％（体积/体积）的乙腈 - 水。 </li><li>使用流速为700微升/分钟，使用梯度洗脱。收集高峰组分。集中零碎蒸发速度真空应用。重要的是要完全干燥之前停止速度的真空。 </li><li>在不同的时间点通过合作获得相关峰植入期URSE。使用纯化的肽，示出一致的趋势，在植入过程中，通过自动Edman测序鉴定。 </li><li>自动Edman测序的肽：确定通过自动Edman降解法使用PROCISE微定的纯化的肽的N-末端序列。加载样品，聚凝胺处理的玻璃纤维过滤器。下一个步骤是通过色谱法在一个用C 18柱（PTH C-18，2.1×200毫米）22识别酰苯硫脲氨基酸（PTH的Xaa）。后得到的序列，它可以被识别由高炉软件使用SWISS-Prot数据库。 </li></ol>

<ol>
	<li>Hollister, 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=Porous+scaffold+design+for+tissue+engineering.">Porous scaffold design for tissue engineering.</a> Nat. Mater. 4, 518-524 (2005).</li><li>Ryan, G., Pandit, A., Apatsidis, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+methods+of+porous+metals+for+use+in+orthopaedic+applications.">Fabrication methods of porous metals for use in orthopaedic applications.</a> Biomaterials. 27, 2651-2670 (2006).</li><li>Schultz, P., Vautier, D., Charpiot, A., Lavalle, P., Debry, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+tracheal+prostheses+made+of+porous+titanium:+a+study+on+sheep.">Development of tracheal prostheses made of porous titanium: a study on sheep.</a> European Archives of Oto-Rhino-Laryngology. 264, 433-438 (2007).</li><li>Janssen, L. 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=Laryngotracheal+reconstruction+with+porous+titanium+in+rabbits:+are+vascular+carriers+and+mucosal+grafts+really+necessary.">Laryngotracheal reconstruction with porous titanium in rabbits: are vascular carriers and mucosal grafts really necessary.</a> Journal of Tissue Engineering and Regenerative. 4, 395-403 (2010).</li><li>Li, J. 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+ingrowth+in+porous+titanium+implants+produced+by+3D+fiber+deposition.">Bone ingrowth in porous titanium implants produced by 3D fiber deposition.</a> Biomaterials. 28, 2810-2820 (2007).</li><li>Schultz, 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=Polyelectrolyte+multilayers+functionalized+by+a+synthetic+analogue+of+an+anti-inflammatory+peptide,+alpha-MSH,+for+coating+a+tracheal+prosthesis.">Polyelectrolyte multilayers functionalized by a synthetic analogue of an anti-inflammatory peptide, alpha-MSH, for coating a tracheal prosthesis.</a> Biomaterials. 26, 2621-2630 (2005).</li><li>M&#252;ller, 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=VEGF-Functionalized+Polyelectrolyte+Multilayers+as+Proangiogenic+Prosthetic+Coatings.">VEGF-Functionalized Polyelectrolyte Multilayers as Proangiogenic Prosthetic Coatings.</a> Advanced Functional Materials. 18, 1767-1775 (2008).</li><li>Mills, R. J., Frith, J. E., Hudson, J. E., Cooper-White, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Geometric+Challenges+on+Cell+Migration.">Effect of Geometric Challenges on Cell Migration.</a> Tissue Engineering Part C-Methods. 17, 999-1010 (2011).</li><li>O'Brien, F. J., Harley, B. A., Yannas, I. V., Gibson, L. J. <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+pore+size+on+cell+adhesion+in+collagen-GAG+scaffolds.">The effect of pore size on cell adhesion in collagen-GAG scaffolds.</a> Biomaterials. 26, 433-441 (2005).</li><li>Karageorgiou, V., Kaplan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porosity+of+3D+biomaterial+scaffolds+and+osteogenesis.">Porosity of 3D biomaterial scaffolds and osteogenesis.</a> Biomaterials. 26, 5474-5491 (2005).</li><li>Budyanto, L., Goh, Y. Q., Ooi, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+porous+poly(L-lactide)+(PLLA)+scaffolds+for+tissue+engineering+using+liquid+-+liquid+phase+separation+and+freeze+extraction.">Fabrication of porous poly(L-lactide) (PLLA) scaffolds for tissue engineering using liquid - liquid phase separation and freeze extraction.</a> J. Mater. Sci. Mater. Med. 20, 105-111 (2009).</li><li>Kim, H. J., Huh, D., Hamilton, G., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gut-on-a-chip+inhabited+by+microbial+flora+that+experiences+intestinal+peristalsis-like+motions+and+flow.">Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow.</a> Lab Chip. 12, 2165-2174 (2012).</li><li>Huh, 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=Reconstituting+Organ-Level+Lung+Functions+on+a+Chip.">Reconstituting Organ-Level Lung Functions on a Chip.</a> Science. 328, 1662-1668 (2010).</li><li>Chaubaroux, 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=Collagen-Based+Fibrillar+Multilayer+Films+Cross-Linked+by+a+Natural+Agent.">Collagen-Based Fibrillar Multilayer Films Cross-Linked by a Natural Agent.</a> Biomacromolecules. 13, 2128-2135 (2012).</li><li>Huang, Y., Siewe, M., Madihally, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+spatial+architecture+on+cellular+colonization.">Effect of spatial architecture on cellular colonization.</a> Biotechnology and Bioengineering. 93, 64-75 (2006).</li><li>Kirkpatrick, C. J., Fuchs, S., Unger, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+systems+for+vascularization+-+Learning+from+nature.">Co-culture systems for vascularization - Learning from nature.</a> Advanced Drug Delivery Reviews. 63, 291-299 (2011).</li><li>Lavalle, 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=Dynamic+Aspects+of+Films+Prepared+by+a+Sequential+Deposition+of+Species:+Perspectives+for+Smart+and+Responsive+Materials.">Dynamic Aspects of Films Prepared by a Sequential Deposition of Species: Perspectives for Smart and Responsive Materials.</a> Advanced Materials. 23, 1191-1221 (2011).</li><li>Zhang, 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=Serum+concentration+of+chromogranin+A+at+admission:+An+early+biomarker+of+severity+in+critically+ill+patients.">Serum concentration of chromogranin A at admission: An early biomarker of severity in critically ill patients.</a> Annals of Medicine. 41, 38-44 (2009).</li><li>Goh, Y., Ooi, C. <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+porous+poly(l+-lactide)+scaffolds+using+solid+-+liquid+phase+separation.">Fabrication and characterization of porous poly(l -lactide) scaffolds using solid - liquid phase separation.</a> Journal of Materials Science: Materials in Medicine. 19, 2445-2452 (2008).</li><li>Vrana, N. 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=Modification+of+macroporous+titanium+tracheal+implants+with+biodegradable+structures:+Tracking+in+vivo+integration+for+determination+of+optimal+in+situ+epithelialization+conditions.">Modification of macroporous titanium tracheal implants with biodegradable structures: Tracking in vivo integration for determination of optimal in situ epithelialization conditions.</a> Biotechnology and Bioengineering. 109, 2134-2146 (2012).</li><li>Dupret-Bories, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+surgical+protocol+for+implantation+of+tracheal+prostheses+in+sheep.">Development of surgical protocol for implantation of tracheal prostheses in sheep.</a> J. Rehabil. Res. Dev. 48, 851-864 (2011).</li><li>Gasnier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+location+of+post-translational+modifications+on+chromogranin+B+from+bovine+adrenal+medullary+chromaffin+granules.">Characterization and location of post-translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules.</a> Proteomics. 4, 1789-1801 (2004).</li><li>Vrana, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+Titanium/Biodegradable+Polymer+Implants+with+an+Hierarchical+Pore+Structure+as+a+Means+to+Control+Selective+Cell+Movement.">Hybrid Titanium/Biodegradable Polymer Implants with an Hierarchical Pore Structure as a Means to Control Selective Cell Movement.</a> PLoS ONE. 6, e20480 (2011).</li><li>Nakatsu, M. N., Davis, J., Hughes, C. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Fibrin+Gel+Bead+Assay+for+the+Study+of+Angiogenesis.">Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis.</a> J. Vis. Exp. (3), e186 (2007).</li><li>Ganguly, A., Zhang, H., Sharma, R., Parsons, S., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Human+Umbilical+Vein+Endothelial+Cells+and+Their+Use+in+the+Study+of+Neutrophil+Transmigration+Under+Flow+Conditions.">Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions.</a> J. Vis. Exp. (66), e4032 (2012).</li><li>Dong, C. -. 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=Photomediated+crosslinking+of+C6-cinnamate+derivatized+type+I+collagen.">Photomediated crosslinking of C6-cinnamate derivatized type I collagen.</a> Biomaterials. 26, 4041-4049 (2005).</li></ol>

NE弗拉纳雇员的Protip SAS。

孔隙梯度接口组织工程中是重要的工具，这里描述的系统可以单独使用，或与金属植入物一起使用，以形成孔梯度，来研究细胞迁移。该系统并不需要任何额外的设置或除了化学通风柜处理有机溶剂的以外的额外的设备，因此它可以被应用在生物实验室中。可以使用类似的聚合物，如聚（乙醇酸）（PGA），聚（乳酸 - 乙醇酸）共聚物（PLGA），聚（己内酯）（PCL），有轻微的修改。也可以用其他的...

目前可用的金属植入物是适合承载的应用程序，但他们的非降解必须设计保证了强劲的接口与周围组织的其中1个。通过提供结构，促进细胞生长和定植在体内的金属植入物的寿命可以延长。公开多孔金属植入物有前途的组织界面工程材料，也为确保良好的殖民化的植入物。他们一直在积极作为骨科植入物及气管植入3-5。但是，仍然存在一些问题需要解决，如细胞的运动精确地控制孔隙区域。不控制这个过程可能会导致不完全的殖民统治，在一端，并在其他的再狭窄。这些植入物的进一步官能化是必要的，为实现更高的功能，如生长因子，送货，直接血管和不同的细胞类型6-8的同步运动。对于气管植入物，植入血管组织的殖民化是可取的，这一点是至关重要的。然而，不受控制的组织内生长气管管腔是不希望的，因为它降低植入物通畅。 控制细胞运动的可能性之一是尺寸排阻。知道的靶细胞和与一个给定的合成聚合物的能力的大小，有可能开发的孔可有效地确定细胞运动的深度的梯度。例如，通过创建一个孔结构是足够大，可以实现结缔组织细胞如成纤维细胞腔外，但足够小（小于10μm），以防止其移动腔内定植的管状植入物有效控制有关条目。 从可用孔隙创建方法，如冷冻dryi纳克，颗粒浸出，气体发泡9,10;最简单的方法以适应快速形成的孔隙梯度少量必要的设备是冷冻萃取11。在该方法中，聚合物溶液的有机溶剂和水的二元混合物中冻结。之后，将溶剂交换通过预冷的可混溶的液体（如乙醇）萃取。冻结和提取条件确定的孔的形状和大小，和如果提取的提取液的运动可控制的方式进行的，孔的大小和形状可以是定向调制。 第二步是多细胞组织形成不同类型的细胞，以控制它们的相互作用多孔之间的壁垒。不同的细胞类型，根据他们的要求12,13不同的微环境的可用性，这也是必要的。气管是一个管状器官连接喉与支气管（一）它有一个内在的假复层纤毛上皮衬里interdispersed产生粘液的杯状细胞。的三维结构和气管稳定保持在C环的形状的软骨。因此，在一个人工气管内，应该有一个定义的结缔组织和睫状体上皮细胞层之间的交界处。的结缔组织部分的三维结构是必要的，上皮细胞的迁移需要基底膜状的表面，以实现定向运动和关闭伤口。聚电解质多层膜（PEM）等是一个可能的选项来获得基底膜模仿。层 - 层的方法（层层）是一种多功能的过程中获得薄的和功能性的表面涂层。它是基于静电相互作用的两个带相反电荷的聚电解质和积聚在以连续的方式，获得纳米级的表面涂层，其属性可以是多种多样的，通过简单地改变的变量，如聚电解质的种类，pH值，层的数目，此外，覆盖层，交联等，其中的LbL方法的主要优点是它能够以符合下面的衬底的地形。因此，在受控的条件下，该方法也可以被用来获得多孔结构的表面覆盖率。如果胶原被用作聚电解质，它有可能取得的nanofibrillar结构，可以模仿基膜的表面。钛的疏水性使这种结构的发展和fibrillarity可以保存在厚厚的涂料14。这种方式附着和细胞的表面上的运动也可以被控制。通过依次使用冷冻提取和层层薄膜包衣，在细胞的运动，可以控制横向，纵向和圆周方向的结构，可以通过以下方式获得15。 这里，我们描述了两个新的修改的方法，利用其疏水性的行为，它可以是钛种植体扩展到修改的各种多孔植入物：ⅰ）形成的微孔梯度内的大孔与疏水性的，合成的聚合物，ⅱ）形成一个厚厚的支持细胞生长和聚电解质多层膜的衣料形成的植入物表面上的聚合物薄膜层的钛植入物。这些方法可以顺序地或单独地使用。他们提供结构确保控制迁移和空间组织的多细胞组织不同类型的细胞16,17。气管的具体情况下，植入物所期望的结果将是由纤维血管组织内没有再狭窄和纤毛上皮细胞上的聚电解质多层膜的内衬形成微孔梯度定植。 控制整合植入的方法之一是做小的外科手术期间，他们的主机集成在原地 。为了能够吨ö决定干预的定时，重要的是有信息的植入物上的全身效应。 C-反应蛋白（CRP）已被用于监测感染和炎症反应在临床设置。嗜铬粒蛋白A（CGA）也可以以类似的方式使用，并可能提供更准确的结果，观察到的水平，炎症18。观察金属植入物在体内的集成作为一种可能的方式中，我们提出了一种植入物的全身效应的表征动物的血液样品，用高压液相色谱法（HPLC）和随后的蛋白质测序连续监测过程。拟订这种方法可以用来逃避定期终点组织学分析。金属植入物的组织学切割是一个长期的，繁琐的和昂贵的过程，可以只在特定时间点进行。因为这个原因，精心设计提供强大的信息植入健康的血液​​测试最近的欧盟关于动物实验的规则所规定的，是可能的路由，以减少动物实验。 这里介绍的方法可用于提高性能的金属植入物，通过官能化或监测现有的植入物的另一种方式。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Dioxane</td>
 <td>Sigma-Aldrich</td>
 <td>360481</td>
 <td>Toxic material, Strictly under chemical hood</td>
 </tr>
 <tr>
 <td>PLLA 
 i. Poly(L-lactide) inherent viscosity ~0.5 dl/g 
 ii. Poly(L-lactide) inherent viscosity ~2.0 dl/g 
 </td>
 <td>Sigma-Aldrich</td>
 <td>94829, 81273</td>
 <td>The choice of molecular weight and inherent viscosity is application dependent.</td>
 </tr>
 <tr>
 <td>PRONOVA UP LVG (Sodium Alginate)</td>
 <td>Novamatrix</td>
 <td>4200006</td>
 <td>Low viscosity(20-200 mPa.s)</td>
 </tr>
 <tr>
 <td>Collagen type I (Bovine)</td>
 <td>Symatese</td>
 <td>CBPE2US100</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen/Strep, Fungizone </td>
 <td>Promocell</td>
 <td>C42020</td>
 <td></td>
 </tr>
 <tr>
 <td>Genipin</td>
 <td>Wako</td>
 <td>0703021</td>
 <td></td>
 </tr>
 <tr>
 <td>Silicon nitride probes with aspring constant of 0.03 N.m-1. </td>
 <td>Bruker </td>
 <td>MSCT </td>
 <td></td>
 </tr>
 <tr>
 <td>Trifluoroacetic acid for HPLC ,&ge;99.0% </td>
 <td>Sigma-Aldrich</td>
 <td>302031</td>
 <td>Hazardous Material, Please follow MSDS carefully</td>
 </tr>
 <tr>
 <td>Acetonitrile, for HPLC ,&ge;99.9% </td>
 <td>Sigma-Aldrich</td>
 <td>34998</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcein-AM</td>
 <td>Invitrogen</td>
 <td>C3100MP </td>
 <td></td>
 </tr>
 <tr>
 <td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling </td>
 <td>Sigma-Aldrich</td>
 <td>PKH26GL</td>
 <td></td>
 </tr>
 <tr>
 <td>Rabbit C-Reactive Protein (CRP) ELISA kit</td>
 <td>Genway Bio</td>
 <td>GWB-9BF960 </td>
 <td></td>
 </tr>
 <tr>
 <td>DMSO, Bioreagent, &ge;99.7% </td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Multimode Nanoscope IV Atomic Force microscope</td>
 <td>Bruker</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Procise microsequencer </td>
 <td>Applied Biosystems </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ultima 3000 HPLC system </td>
 <td>Dionex</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Scanning Electron Microscope Hitachi TM 100</td>
 <td>Hitachi </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Confocal Scanning Laser Microscope Zeiss LSM 510 </td>
 <td>Zeiss </td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
 </table>
Table 1. List of Materials and Reagents.

multi-scale modification of metallic implants with pore gradients, polyelectrolytes and their indirect monitoring in vivo

金属植入物，特别是钛植入物，在临床应用中被广泛使​​用。这些植入物在组织中的组织生长和集成是成功的临床结果的重要参数。为了提高组织整合，多孔金属植入物已被开发出来。开口孔隙率的泡沫金属是非常有利的，因为可官能化的孔区域，而不会影响整个结构的机械性能。在这里，我们描述了这样的修改，使用基于钛微球多孔钛种植体。通过使用固有的物理特性，如疏水性的钛，未能取得基于微珠的金属植入物内，并在同一时间有基底膜模仿基于亲水性的，天然的聚合物的疏水性的孔隙梯度。三维孔隙梯度形成的合成聚合物，例如聚-L-乳酸（PLLA）冷冻提取方法。 2D nanofibrillar河畔面形成有通过使用胶原/藻酸盐，然后用天然的交联剂交联步骤（京尼平）。此nanofibrillar电影始建层层沉积法（LBL）两种带相反电荷的分子，胶原蛋白和海藻酸。最后，在不同的区域可以容纳不同类型的细胞，因为这是所必需的许多多细胞组织的植入物，可以通过以下方式获得。通过，这样一来，可以控制由不同的细胞类型的细胞在不同方向的运动。气管再生为特定的情况下，描述了这样的系统，但它可以被修改为其它靶器官。细胞迁移和可能的方法来创建不同孔径梯度分析阐述。这种植入物的分析的下一个步骤是在植入后的表征。然而，金属植入物的组织学分析是一个漫长而繁琐的过程，从而为监控主机的金属植入物的反应在体内是一个基于CGA和不同血液中的蛋白质监测延误保障的方法也作了说明。这些方法可以用于开发量身定制在体外迁移和定植测试的，也可用于官能化的金属植入物在体内无组织学分析。

孔梯度的形成通过改变聚乳酸溶液的浓度，它是可能的植入物上的腔外侧的孔的大小来控制。孔隙的大小和形状显着影响的钛植入物的存在下（ 图1a和1b）。孔径大小范围为40-100微米，利用较低的浓度导致在更小的孔隙。然而，在所述管腔内侧面孔径大小是受限制的提取，约9微米23，成纤维细胞的平均尺寸小于。通过添加，在室温下温育步骤的?...

Watch this Scientific Journal Video about Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo at JoVE.com

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

在这段视频中，我们将演示多孔金属植入物的改性技术，以提高它们的功能和控制细胞迁移。这些技术包括孔梯度发展，控制细胞的移动3D和生产基底膜模仿在2-D控制细胞运动。此外，描述一个基于HPLC的监测方法，通过分析血液中的蛋白质被检体内的植入物的整合。

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 ...

multi-scale-modification-metallic-implants-with-pore-gradients

Research

JoVE Journal

Bioengineering

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

10.8K 次观看.  INSERM. 在这段视频中，我们将演示多孔金属植入物的改性技术，以提高它们的功能和控制细胞迁移。这些技术包括孔梯度发展，控制细胞的移动3D和生产基底膜模仿在2-D控制细胞运动。此外，描述一个基于HPLC的监测方法，通过分析血液中的蛋白质被检体内的植入物的整合。

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

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

监测与固态纳米孔蛋白质吸附

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

科研

生物工程

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

生物医学植入物的石墨涂料

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

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 ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

可扩展的，金属的高宽比纳米复合材料在生物液体中产生

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

基于角蛋白纳米纤维生物医学工程合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

的简便和环保路线制造聚乳酸支架与分级孔径

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

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 ...