These studies were funded by National Medical Research Council Clinician Scientist - Individual Research Grant (CS-IRG) NMRC/CIRG/1372/2013 and NMRC EDG/0022/2008.

1.海藻酸钠溶液制备<ol><li>溶解200毫克藻酸钠（未照射），或者400毫克8兆拉德的在10ml双蒸水照射海藻酸钠振摇1小时的非辐射藻酸盐和辐照藻15分钟。过夜储存在4℃的藻酸盐溶液。滤波器海藻酸钠微珠制作之前无菌0.2微米的注射器过滤的藻酸盐溶液。 </li></ol> 2.海藻酸钠微珠制作<ol><li>消毒静电珠发生器和注射器泵用70％乙醇，并放置在一类二生物安全柜（ 图1）。 </li><li>放置一个玻璃盆珠发生器内磁力搅拌棒。 </li><li>设置珠发生器盆高于9厘米的臂电极。 </li><li>珠发生器的电极电缆连接到手臂电极的滚花螺钉2，倒加入80ml SRCL 2溶液进盆地。 </li><li>负载5毫升0.2微米过滤藻酸盐溶液到注射器和橡胶管。橡胶管连接到臂电极后，以5毫升/小时打开注射泵2分钟以排出管道内的空气，并提供藻酸盐溶液到喷嘴的前端。关掉注射器泵。 </li><li>接下来，切换对封装，然后，将注射泵开始微珠生成。设定为5毫升/小时的藻流速和在5.8千伏的封装上的电压。丢弃前两分钟（或泵出注射器的初始0.5毫升藻酸盐溶液）期间产生的微珠，因为这些微珠趋于不规则尺寸和形状。 </li><li>收集在0.2M的氯化锶溶液随后微珠。同时关闭抽藻酸盐溶液的预先计划体积后注射泵和封装器（按该顺序）。重复此微珠制作后续批次。项目建成后，转F中的注射泵第一，其次是封装。 </li><li>在4℃下储存的微珠在20毫升的0.2M氯化锶溶液过夜以完成交联和稳定的凝胶。 </li></ol> 3.海藻酸钠微球的尺寸测量<ol><li>收集0.5海藻酸钠微珠毫升用塑料移液管，并放置在载玻片上。查看在光学显微镜下微珠在10倍的放大倍率。就拿微珠的十张图片，用显微镜CCD摄像头。在2048点¯x1536的分辨率保存在TIFF格式比例尺（500微米）的图像。 </li><li>在ImageJ的使用长度工具，测量微珠和比例尺（ 图2）的大小。从像素转换微珠长度微米。 <ol><li>点击行工具和绘制穿过藻酸盐珠的中间线。 </li><li>点击菜单栏上的"分析"，然后选择"测量"。会出现一个弹出窗口。 </li><li&gt;重复步骤3.2.1-3.2.2测量图像中的所有藻酸盐珠。测量图像的比例尺。 </li><li>比例尺×500微米的胶珠/长度长度:通过使用公式转换藻酸盐珠的实际长度的直径。例如，1.420（直径ImageJ的测量）/ 2.657（由ImageJ的衡量标尺长度）×500微米= 267微米。 </li></ol></li><li>考虑100微珠的平均尺寸（平均值±标准偏差），为微珠每批代表尺寸。 </li></ol> 4.消毒<ol><li>收集使用100微米的尼龙过滤器微珠并用双蒸水洗涤珠。 </li><li>使用刮刀，传送由0.1mL藻酸盐溶液进行的所有微珠入2ml微量离心管中，用纱布覆盖，以防止干燥。 </li><li>最后，通过使用液体模式高压灭菌消毒的微珠（115℃，15分钟），或按照以manufacturer的规格。加1.5升蒸馏水到室，以防止干燥的珠子。 </li></ol> 5.鱼精蛋白和肝素涂层<ol><li>内的BSL-2罩，孵育1毫升的2毫克/毫升鱼精蛋白溶液在室温下1小时（用0.2μm注射器过滤灭菌的）无菌微珠。 </li><li>孵化微珠1小时（步骤5.1）后，收集150微升的微型二喹啉甲酸（microBCA）测试（第6）鱼精蛋白的解决方案。 </li><li>用双蒸水洗净，鱼精蛋白涂层微珠的两倍。降速使用台式离心机以200×g离心在室温下3分钟。离心后，吸用注射器水中。 </li><li>孵育鱼精蛋白涂覆的微珠用1ml的0.5毫克/毫升肝素的溶液30分钟，以创建聚电解质复合物（PEC）（使用0.2微米的注射器过滤灭菌的）。 </li><li>孵育涂鱼精蛋白微˚F后或30分钟（步骤5.4），收集400微升肝素溶液，以确定肝素含量（第7条）。 </li><li>温育后，通过用双蒸水洗涤两次洗掉从胸肌未结合的肝素。 </li></ol> 6.鱼精蛋白含量<ol><li>根据制造商的说明进行microBCA测试。简要地说，添加150微升的鱼精蛋白溶液（收集前，用微珠孵育后）到96孔板中。添加150微升microBCA工作溶液。 </li><li>使用白蛋白溶液（0,0.5，1，2，5，10，20，40和200微克/毫升）作为校准标准。 </li><li>孵育在60℃下进行60分钟该混合物。测量用在562nm处用分光光度计的吸光度。 </li><li>使用标准曲线来确定根据制造商的说明每个未知样品的鱼精蛋白的浓度。 </li><li>通过在减去鱼精蛋白的总量确定的微珠的鱼精蛋白含量从精蛋白的残留在涂布液（用微珠孵育后）的量涂布液（用微珠孵育之前）。 </li></ol> 7.肝素内容<ol><li>通过将4毫克甲苯胺蓝和在0.01N盐酸20毫克氯化钠制备10毫升工作溶液。 </li><li>添加400微升样品（来自步骤5.5），以工作溶液以2:3的比例为30秒和旋涡。 </li><li>添加600μl的正己烷（等效体积的工作试剂溶液）并涡旋混合物以提取甲苯胺蓝肝素络合物。 </li><li>抽吸将200μl的水相用注射器相分离之后。 </li><li>测量使用分光光度计在631纳米包含在水相中的未提取的甲苯胺蓝的量。 </li><li>准备0-20微克/毫升肝素标准的解决方案。 </li><li>绘制每个标准肝素对肝素浓度的631 nm的读数微克/立方米湖使用标准曲线来确定各样品的肝素浓度。 </li></ol> 8层 - 层结构的共焦成像<ol><li>制造鱼精蛋白，肝素和NELL-1 / BMP-2的荧光类似物的CF-405精蛋白（蓝色），CF 594肝素（红色）和FITC标记NELL-1 / FITC标记根据（绿色），BMP-2 +肝素+精蛋白制造商的技术数据表。 </li><li>外套100微克微珠用300μl荧光类似物（如在5.3-5.6中所述涂覆法）的CF-405精蛋白（蓝色）（2毫克/毫升，1小时温育），CF 594肝素（红色）（0.5毫克/毫升， 30分钟）和FITC标记NELL-1 / FITC标记（绿色）的BMP-2（1.5毫克/毫升，过夜）。用蒸馏水冲洗微珠两次，以消除未结合的荧光鱼精蛋白，肝素和NELL-1 / BMP-2。 </li><li>通过在10倍的放大倍率使用共焦显微镜观察该层的分层结构（ 图3）。7 </li></ol> 9. BMP-2的和NELL-1吸收和释放<ol><li>装载13.3微升1.5毫克/毫升100微克PEC的BMP-2或NELL-1解决方案。在4℃下30 rpm振摇10小时孵育PEC。 </li><li>在37℃下恒定振荡（30rpm下）沉浸在1ml磷酸盐缓冲盐水（PBS）的微珠。 </li><li>收集1毫升上清液和后1,3，6,10和14天用1ml的PBS替换。 </li><li>评估的摄取和使用根据制造商的协议ELISA方法释放的BMP-2的效率。评估的摄取和使用根据制造商的协议的羧基苯甲酰喹啉-2-甲醛（CBQCA）蛋白测定法释放NELL-1的效率。 </li><li>在时间（t）确定累积释放: 在时间（t）=释放累积释放的时间（t）+前一版本在时间（t-1）。 </li><li>绘制BMP-2和NELL-1的累积释放对时间。 </li></ol> 10. 在体外生物活性NELL-1的注:NELL-1 PEC释放的生物活性，通过测定其增加对兔骨髓干细胞（rBMSC）碱性磷酸酶（ALP）的表达能力进行评估。 <ol><li>种子每孔20,000个rBMSCs在24孔板中并允许它们在37℃和5％CO 2的胎牛血清（FBS），1毫升的Dulbecco氏改良Eagle氏培养基（DMEM）+ 10％的增长一天。 </li><li> 24小时后，更换用1ml成骨培养基（DMEM补充有10％胎牛血清，2％青霉素链霉素，50微克/毫升抗坏血酸，10毫摩尔/升的β-甘油磷酸的培养基中，10 -8 mol / L的地塞米松）7天，在37℃和5％的CO 2。 </li><li>将300微克PEC-NELL-1（步骤8.2）和PEC细胞培养插入内（TC插入），以保持胸肌从细胞（这避免了成骨细胞培养基变化期间PEC微珠的冲洗）分开。地方TC插入24以及PL吃了14天。 </li><li>每三天一次，吸将1ml成骨培养基中通过将针TC插入外，并用1ml新鲜成骨培养基的替换。 </li><li>后7和14天的潜伏期，确定与在按照试剂盒生产商的方案的ALP测定试剂盒ALP活性。 <ol><li>裂解细胞用含有0.1％的TritonX-100，在4℃下进行10分钟的测定缓冲液。刮去使用细胞刮粘附细胞​​。在4℃下孵育细胞悬浮液在搅拌下至少60分钟。 </li><li>离心细胞悬浮液以2500 xg离心在4℃下10分钟。收集的ALP测定上清。 </li><li>加入50μl连续稀释碱性磷酸酶标溶液的从200到0纳克/毫升至96孔板的孔中。碱性磷酸酶标准最终量是10，5，2.5，1.2，0.6，0.3，0.15，和0纳克/孔。 </li><li>从步骤10.5.2添加上清液（50μl/孔），并稀释与B类稀释uffer。 </li><li>加入50μl的对硝基苯磷酸酯（物pNPP）底物溶液加入到每个孔中。通过轻轻晃动板30秒混合试剂。孵育在黑暗中30分钟该混合物。测量在通过板读数器405处的吸光度。 </li><li>使用校准曲线计算碱性磷酸酶活性。 </li></ol></li><li>确定根据制造商的说明使用microBCA蛋白测定试剂盒蛋白质含量。由蛋白质含量除以碱性磷酸酶活性规范化碱性磷酸酶活性。 </li></ol> 11.细胞活力<ol><li>在37℃下孵育200毫克PEC-NELL-1用1ml的DMEM + 10％FBS的用于3-（4,5-二甲基吡啶-2-基）24小时-2,5-二苯基溴（MTT）检测。 </li><li>种子2000 rBMSCs每孔（在100μl的DMEM含10％FBS）在96孔板中并孵育1天，在37℃，5％ 的 CO 2。 </li><li>用100μlPEC-NELL-1 / PEC提取物代替DMEM + 10％FBS中孵育在37℃，5％ 的 CO 2。 </li><li>后第1天或培养3天，添加10微升5mg / ml的MTT溶液，在37℃，5％的CO 2进一步孵育在黑暗中4小时。 </li><li>加入100微升的DMSO溶液，以每孔以溶解甲晶体。 </li><li>确定在使用酶标仪570nm处的吸光度。 </li><li>计算相对增长率: <img alt="式（1）" src="/files/ftp_upload/54202/54202eq1.jpg" /></li></ol> 12.包装成支架和BMP-2 NELL-1加载<ol><li>使用BSL-2室内的灭菌抹刀磷酸三钙（MPCL-TCP）脚手架 - 包装胸肌成生物可吸收的医用级聚己内酯的孔中。 </li><li>添加1.5毫克/ BMP-2或NELL-1毫升溶液到填充有PEC的MPCL-TCP支架材料和孵育过夜，在4℃。 </li></ol>

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该协议提出了通过层 - 层自组装制备的PEC的方法。的层 - 层结构，使用鱼精蛋白，肝素，BMP-2和NELL-1和共聚焦显微镜的荧光类似物可视化。吸收和释放试验表明，肝素对PEC介导成骨生长因子的吸收和释放。在PEC方法的摄取效率是:NELL-1:86.7±2.7％，BMP-2:70.5±3.1％。在PEC载体具有NELL-1（20％）释放的更好的调制相比，纯表面吸附载体如钙磷灰石颗粒（40-80％）11。 除了 ​​?...

假的发病率已经报道高达10〜45％，在退化性脊柱融合和修订脊柱手术1。以减少脊柱融合和其他重建骨手术，骨生长因子如BMP-2，内尔-1 1和血小板衍生的生长因子（PDGF）已被引入，以促进从头成骨过程中假关节的速度。其中，BMP-2是脊柱融合2的热门选择。虽然BMP-2的诱导和促进新骨形成的效力已被很好地建立3;临床显著并发症，如异位骨形成，血清肿和血肿形成，炎症反应，神经根，椎体骨溶解和逆行射精继续关注的问题，由于使用的4,5超生理金额。 因此，降低的BMP-2的剂量保持在一个相关的策略引诱最小化的副作用。此外，需要有效的载体系统来抑制BMP-2在当代胶原海绵载波系统观察到的突释，并进一步加强这种有效的细胞因子的长期和本地化交货。交替阳离子和阴离子聚电解质的层-层自组装，可以用作建立脚手架基质或植入材料6的表面上的聚电解质复合物的可调谐方法。在这方面，肝素（对于具有所有生物剂的最高负电荷密度已知）已被识别为贪婪地通过静电和肝素结合结构域的多种生长因子结合。的确，肝素已经显示出延长的半衰期，从而使可能的多种生长因子的生物活性。 在此基础上，我们的组适于层 - 层自组装协议来制造基于肝素的聚电解质复合物（PEC），该<html>负载和固定7,8中保留了成骨生长因子的生物活性。藻酸盐微球芯由交联的α-L-古洛糖醛酸盐（G），用二价阳离子钙或锶离子藻酸盐的残基制成。藻酸盐核心是可生物降解的支架基质;该植入后，它是在融合床提供余地骨向内生长吸收。聚-L-赖氨酸（PLL）或鱼精蛋白被用作阳离子层与两个支架矩阵（在这种情况下，藻微珠载体芯）和带负电的肝素隔行;而阴离子肝素层的功能，以稳定和定位加载生长因子。三重层的PEC已经显示出增加的生长因子负载能力在猪模型9。最近，PEC载体已显示出至少20倍的成功减少在大鼠10和脊骨融合8的猪模型的BMP-2的有效剂量。 ntent"&gt;在这里，用BMP-2作为模型骨生长因子，我们报告脊柱融合增强生长因子交付和其他骨重建手术制造佩奇方法。

<table border="0" cellpadding="0" cellspacing="0" width="981">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Life Science Acrodisc 25mm Syring Filter w/0.2 &#181;m Supor&#160; Membrane</td>
			<td style="width:167px;">PALL&#160;</td>
			<td style="width:79px;">PN4612</td>
			<td style="width:311px;">Sterile protamine, 
			&#160;heparin solution by ultrafiltration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">24 well plate</td>
			<td style="width:167px;">Cell Star&#160;</td>
			<td style="width:79px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">96 well plate Nuclon Delta Surface</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">167008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), MTT</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">M5655</td>
			<td>Measure cytotoxicity of PEC-NELL-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Acetone</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:79px;">A/0600/17</td>
			<td style="width:311px;">Precipitate CF-405 
			Labelled protamine&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Alamar Blue</td>
			<td style="width:167px;">Invitrogen, Life Technologies</td>
			<td style="width:79px;">DAL 1025</td>
			<td>Measure cytotoxicity of PEC-BMP-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Alkaline Phosphatase Assay (ALP) assay kit</td>
			<td style="width:167px;">Anaspec</td>
			<td style="width:79px;">AS-72146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Ammonium Chloride</td>
			<td style="width:167px;">Merck</td>
			<td style="width:79px;">Art 1145</td>
			<td>Stop reagent in FITC labelling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Anhydrous Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:167px;">Invitrogen, Life Technologies</td>
			<td style="width:79px;">D12345</td>
			<td style="width:311px;">Solvent for fluorescent isothiocyanate I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;"></td>
			<td style="width:311px;">Dissolve&#160; formazan&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Autoclave</td>
			<td style="width:167px;">Hirayama</td>
			<td style="width:79px;">HU-110</td>
			<td>Sterilize alginate beads by steam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beta-glycerophosphate</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">G9422</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:425px;">BMP-2 (Infuse Bone Graft Large II Kit)&#160;</td>
			<td style="width:167px;">Medtronic Sofarmor Danek, Memphis TN, USA</td>
			<td style="width:79px;">7510800</td>
			<td style="width:311px;">Osteogenic Growth&#160; Factor, 
			&#160;dialysis is needed to remove stabilizer component that interferes with FITC coupling</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:425px;">Carboxybenzoyl quinoline-2-Carboxaldehyde (CBQCA)&#160;</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">A-6222</td>
			<td>To quantify NELL-1 protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Cell Strainer (100&#181;m)</td>
			<td style="width:167px;">BD Science</td>
			<td style="width:79px;">352360</td>
			<td>Hold PEC for ALP assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Cell Scraper 290mm Bladewide 20mm</td>
			<td style="width:167px;">SPL Life Science&#160;</td>
			<td style="width:79px;">90030</td>
			<td>Detach the cell from 24 well plate&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">CF 405S, Succinimidyl Ester</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">SCJ4600013</td>
			<td>Blue fluorescent dye for protamine labelling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">CF 594, Hydrazide</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">SCJ4600031</td>
			<td>Deep red fluorescent dye for heparin labelling&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Centrifuge</td>
			<td style="width:167px;">Beckman Coulter</td>
			<td style="width:79px;">Microfuge 22R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Confocal Microscope</td>
			<td style="width:167px;">Olympus&#160;</td>
			<td style="width:79px;">FV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Dexamethasone</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">D4902</td>
			<td>Component of osteogenic growth medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Dextran Desalting Columns</td>
			<td style="width:167px;">Pierce (Thermo Scientific)&#160;</td>
			<td style="width:79px;">43230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">DMEM</td>
			<td style="width:167px;">Gibco&#160;</td>
			<td style="width:79px;">12320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">BMP-2 Quantikine ELISA Kit</td>
			<td style="width:167px;">R&#38;D System</td>
			<td style="width:79px;">DBP200</td>
			<td>Determine BMP-2 release</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Fetal Bovine Serum FBS</td>
			<td style="width:167px;">Hyclone</td>
			<td style="width:79px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Fluoescein Isothiocyananate, Isomer I</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">F7250</td>
			<td>Green fluorescent dye for NELL-1 and BMP-2 labelling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">ThinCert Cell Culture Inserts, 
			For 24 Well plates, Sterile</td>
			<td style="width:167px;">Greiner&#160;</td>
			<td style="width:79px;">662630</td>
			<td style="width:311px;">Prevents PEC wash out when 
			&#160;changing osteogenic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Havard Appartus Syringe Pump (11 plus)</td>
			<td style="width:167px;">Havard Apparatus</td>
			<td style="width:79px;">70-2208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">n-Hexane (&#62;99%)</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">139386</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Heparin</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">H3149</td>
			<td style="width:311px;">Binds with osteogenic 
			growth factor with heparin binding domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Hydrochloric acid (37%)</td>
			<td style="width:167px;">Merck</td>
			<td style="width:79px;">100317</td>
			<td>Highly Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Incubator</td>
			<td style="width:167px;">Binder</td>
			<td style="width:79px;">C8150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">MicroBCA Protein Assay kit</td>
			<td style="width:167px;">Thermoscientific</td>
			<td style="width:79px;">23235</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Microplate Reader</td>
			<td style="width:167px;">Tecan</td>
			<td style="width:79px;">Infinite M200</td>
			<td>For ALP and microBCA assays</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">NELL-1</td>
			<td style="width:167px;">Aragen Bioscience Morgan Hill, CA, USA</td>
			<td style="width:79px;">N/A</td>
			<td>Osteogenic growth factor, keep at -80&#730;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:425px;">Nisco cell encapsulator</td>
			<td style="width:167px;">Nisco Engineering Inc</td>
			<td style="width:79px;">Encapsulation unit VAR V1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Fluorescent Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:79px;">IX71</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">mPCL-TCP Scaffold (Pore size is 1.3mm)</td>
			<td style="width:167px;">Osteopore</td>
			<td style="width:79px;">PCL-TCP 0/90</td>
			<td>Hold PEC for in vivo study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Penicillin-Streptomycin 10,000 unit/ml, 100ml</td>
			<td style="width:167px;">Hyclone Cell Culture</td>
			<td style="width:79px;">SV30010</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">10X Phosphate Buffered Saline (PBS)&#160;</td>
			<td style="width:167px;">Vivantis</td>
			<td style="width:79px;">PB0344-1L</td>
			<td>10x Solution, Ultra Pure Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Poly-L-Lysine MW 15,000-30,000</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">P2568</td>
			<td>Polycation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Protamine Sulfate salt, from Salmon</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">P4020</td>
			<td>Polycation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Shaker</td>
			<td style="width:167px;">Labnet</td>
			<td style="width:79px;">S2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Snakeskin Dialysis Tubing 3,500 MWCO 22mm x 35 feet</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">68035</td>
			<td>Remove unreacted FITC by dialysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Sodium Chloride</td>
			<td style="width:167px;">Merck</td>
			<td style="width:79px;">1.06404.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Sodium Hydroxide</td>
			<td style="width:167px;">Qrec</td>
			<td style="width:79px;">S5158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Sodium Bicarbonate</td>
			<td style="width:167px;">US Biological</td>
			<td style="width:79px;">S4000</td>
			<td>Buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Sodium carbonate</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">S7795-500G</td>
			<td>Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Strontium Chloride Hexahydrate</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">255521</td>
			<td>Crosslinker for alginate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Spatula</td>
			<td style="width:167px;">3dia</td>
			<td style="width:79px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">5ml syringe</td>
			<td style="width:167px;">Terumo</td>
			<td style="width:79px;">140425R</td>
			<td style="width:311px;">Diameter of syringe 
			affects the flow rate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">75cm2 Cell Culture Flask Canted Neck</td>
			<td style="width:167px;">Corning</td>
			<td style="width:79px;">730720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Toluidine Blue&#160;</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">52040</td>
			<td>Heparin assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Trypsin 1X</td>
			<td style="width:167px;">Hyclone Cell Culture</td>
			<td style="width:79px;">SH30042.01</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:425px;">Sodium alginate</td>
			<td style="width:167px;">Novamatrix (FMC Biopolymer, Princeton, NJ)</td>
			<td style="width:79px;">Pronova UPMVG</td>
			<td>Core material of microbeads</td>
		</tr>
	</tbody>
</table>

polyelectrolyte complex for heparin binding domain osteogenic growth factor delivery

在骨重建手术，生长因子超生理金额经验装上支架，以促进成功的骨融合。大剂量的高度有效的生物制剂的需要，由于生长因子的不稳定性如快速酶降解的结果，以及在植入物位点定位足够量的生长因子的载体的低效率。因此，该延长的生长因子的稳定性，如BMP-2 / NELL-1，并控制其释放策略实际上​​可以降低其有效剂量，从而减少了需要更大的剂量期间未来骨再生手术。反过来，这将减少副作用和生长因子的成本。自组装胸肌已经制造通过增强的体内稳定性通过肝素结合，并进一步使可能生长因子的生物活性提供的BMP-2 / NELL-1递送的更好的控制。在这里，我们说明了PEC制造这有助于在国内配送简单的过程中重建骨手术的各种生长因子RY。

在我们的载体，鱼精蛋白被选为聚-L-赖氨酸的替代，因为它有类似的化学性质，它是FDA批准作为肝素的解毒剂。光学显微镜结果表明，非照射微珠呈球状的形状，直径为267±14微米。 （0.35毫米喷嘴，流量为5ml /小时和5.8千伏率）。大部分照射微珠是泪滴形状。上照射微珠的轮部测得的直径为212±30微米（0.35毫米喷嘴，流速为4毫升/小时和6千伏率）。 （ 图4）。 ...

Watch this Scientific Journal Video about Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery at JoVE.com

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

polyelectrolyte-complex-for-heparin-binding-domain-osteogenic-growth

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Bioengineering

7.5K 次观看.  National University of Singapore. Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

视频: Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

轴突伸展生长:神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

科研

生物工程

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

界面聚电解质纤维的生物分子在时间控释复合支架

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

多孔硅薄膜作为神经生长因子载体的设计

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

使用 PDMS 的高通量牵引力显微镜揭示了转化生长因子的剂量依赖效应 - 对中皮到中位过渡的影响

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...